Introduction

Neuromuscular junction (NMJ) is a specialized chemical synapse, where axonal action potential is converted into activation of the muscle fiber. In vertebrate NMJs, the main neurotransmitter is acetylcholine (ACh), which is released from synaptic vesicles (SVs) and then activates postsynaptic nicotinic ACh receptors (nAChRs) leading to end plate depolarization. Several tens of SVs undergo exocytosis in response to single nerve stimulus at the mammalian NMJs, this number of ACh quanta significantly exceed that required to trigger an action potential on the sarcolemma [1, 2]. On the one hand, this ensures the reliability of neuromuscular transmission. On the other hand, excessive exocytosis can lead to depletion of SV pool during intense activity, since slow kinetics of vesicle endocytosis cannot keep up with the rate of exocytosis [3, 4]. In addition, excessive ACh release causes high energy expenditure [5] and even premature aging of NMJs [6]. Accordingly, negative feedback loops are vital for adaptation of neuromuscular transmission and limitation of ACh exocytosis. One of these loops is realized through the action on presynaptic receptors of co-transmitter molecules, which, along with ACh, are present in SV [7, 8].

ATP is the main co-transmitter of ACh in the vertebrate NMJs [7,8,9]. Over time, released ATP is enzymatically hydrolyzed to ADP, AMP and eventually adenosine in the synaptic cleft. ATP and ADP modulate neurotransmission via activation of inotropic P2X-or (and) metabotropic P2Y-purinoceptors, whereas adenosine acts though metabotropic adenosine (P1) receptors [8]. ATP/ADP-dependent suppression of ACh release from vertebrate motor nerve terminals appears to be primarily due to activation of Gi protein-coupled P2Y receptors, without marked contribution of P2X receptors [9,10,11,12,13]. At the frog NMJs, ATP reduced ACh release acting via P2Y12 receptors / NADPH oxidase (NOX)/reactive oxygen species (ROS) axis [9, 10, 13]. Generated ROS might inhibit ACh release by affecting the exocytotic protein SNAP-25 [14] or ROS-sensitive signaling molecules and ion channels in the frog NMJs [15, 16]. At the mouse NMJs, P2Y13 receptors mediated adenine nucleotides-dependent presynaptic inhibition of ACh release [11, 17]. This receptor subtype was identified in the presynaptic motor nerve terminals of mouse diaphragm [11]. However, a downstream signaling mechanism of P2Y13 receptor-driven regulation of neurotransmission in the mammalian NMJs remains elusive.

Given the relationship between P2Y13 receptors and lipid trafficking [18,19,20], functioning of these receptors can be dependent on a lipid microenvironment, specifically lipid raft stability. Lipid rafts are abundant in the NMJ membranes [21,22,23,24] and the junctional lipid raft disturbance was observed after short-term muscle disuse [25, 26] and at the early stage in models of amyotrophic lateral sclerosis [27, 28].

In the present study, we tested the hypothesis that NOX-derived ROS is a key element in purinergic regulation of neurotransmitter release at the mouse NMJs. Additionally, the relevance of Ca2+ signaling and lipid microenvironment for purinergic regulation of exocytosis was assessed. We demonstrated that exogenous (ADP) and endogenous purines acting on P2Y13 receptors suppress ACh exocytosis, causing a rapid Ca2+ release from intracellular stores, protein kinase C activation, NOX-mediated generation of ROS. The integrity of lipid raft components, cholesterol and sphingomyelin, is required for P2Y13 receptor-dependent attenuation of SV exocytosis.

Methods

Ethical Approval and Animals

Animal care and experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the EU Directive 2010/63/EU. The experimental protocol was approved by the Local Ethical Committee of Kazan Medical University (Protocol #1/January 25, 2022) and Kazan Federal Scientific Centre (#23/7; May 12, 2023). All efforts were made to minimize animal suffering. BALB/c mice were housed at a 12-h light/12-h dark cycle in ventilated cages at temperature and humidity-controlled environment with free access to food and water. Animals were not intentionally randomized and investigators were not blinded to the nature of chemicals used. After anesthesia of mice (both sexes, 4–5-month-old), diaphragm with phrenic nerve stubs was quickly excised and hemidiaphragm-phrenic nerve preparations were placed into experimental chambers. The hemidiaphragm was attached to Sylgard-coated bottom of the chamber (total volume of 5 ml) and the phrenic nerve stub was loosely drawn into a suction electrode.

Solutions and Chemicals

The nerve-muscle preparations were continuously perfused at 5 ml/min with physiological solution (pH-7.4) containing (in mM): 120NaCl, 5KCl, 2CaCl2, 1MgCl2, 1NaH2PO4, 24NaHCO3, 11 glucose; the solution was saturated with carbogen (5% CO2 and 95% O2 mixture). These reagents were purchased from Sigma. In some experiments, modified physiological solution, containing high K+ (40 mM KCI; NaCI concentration was decreased to keep normal osmolarity, 310 ± 5 mOsm/L) was used. The osmolarity was measured using an OSMOMAT 3000 Osmometer (Gonotec GmbH). In electrophysiological experiments, the muscle fibers were cut transversely (‘cut muscles’) to prevent muscle contractions and to simultaneously maintain the physiological level of quantal release and cable properties [29]. In the cut muscles, recording began after the stabilization of membrane potential (i.e., ⁓ 50–60 min after the cutting procedure). In fluorescent experiments, the contractions were blocked by 15 min pre-exposure to inhibitor of postsynaptic nicotinic ACh receptor antagonist d-tubocurarine (2 µM; Cat. # 5.05145, Sigma).

Application of neutral sphingomyelinase (SMase; 0.01 U/ml; Cat. # S9396; Sigma), which partially disrupts junctional lipid rafts by hydrolyzing membrane sphingomyelin, lasted 15 min [30]. Another lipid raft destabilizing enzyme cholesterol oxidase (ChO, 0.2 U/ml; Cat. # 228,250, Sigma) was also applied for 15 min [31]. To inhibit Gi/o protein, mice were injected by pertussis toxin (PTX; Cat. # 70,323–44-3, Sigma) at dose of 150 μg/kg (in sodium-phosphate buffer) 72 h before the experiments [32]. The following chemicals were used: 10 µM ADP (an agonist of P2Y1-, P2Y12-, P2Y13-receptors; Cat. # 01905, Sigma), 100 µM IDP/inosine 5′-diphosphate sodium salt (a preferential agonist for P2Y13-receptors; Cat. # NI10027, Biosynth), MRS 2211 (an antagonist of P2Y13-receptors, 10 µM; Cat. # 2402, Tocris), 1 mM N-acetylcysteine/NAC (an antioxidant; Cat. # A7250, Sigma), 1200 U/ml catalase (an antioxidant enzyme; Cat. # C9322, Sigma), 1 μM chelerythrine chloride (protein kinase C inhibitor; Cat # C-400, Alomone labs), 10 μM TMB8 (an inhibitor of intracellular calcium mobilization; Cat. # T111, Sigma). MRS 2211, NAC, catalase, chelerythrine and TMB8 were added to the bathing solution 30 min before P2Y agonist administration and remained in perfusion throughout the experiment. All chemicals were dissolved to the final concentrations in working solution immediately before use.

Electrophysiological Recordings

Recording of the postsynaptic end-plate currents (EPCs) and miniature EPCs (MEPCs) were performed using standard two-electrode voltage-clamp technique described previously in details [33, 34]. Briefly, two intracellular sharp borosilicate-glass microelectrodes (tip diameter of ⁓ 1 µm, 4–5 MΩ resistance) filled with 2.5 M KCl were impaled into the junctional region with interelectrode distance of ⁓100 μm. MEPCs and EPCs were detected using Axoclamp 900 A amplifier (Molecular devices, USA) and LA 2 digital I/O board (Rudnev-Shelyev, Moscow, Russia). In the case of EPC recordings, the value of postsynaptic membrane potential was maintained at − 45 mV (leak currents were in the range of 10–20 nA). EPCs were evoked by supra-threshold stimuli (duration of 0.1 ms) at 0.05 Hz. The stimuli were applied to the phrenic nerve via a suction electrode connected to DS3 stimulator (Digitimer Ltd., UK). Recordings of MEPCs were performed on intact muscles with the resting membrane potentials maintained at − 60 mV; signal-to-noise ratio was > 5:1 and threshold for the MEPC detection was set at a level of 2 nA. In case of MEPC recordings, no less than 8 muscle fibers were studied in each animal. Amplitudes, 20–80% rise time, and half decay time (from the peak to 50%) of MEPCs and EPCs were calculated. The signals were digitized at 50 kHz and analyzed off-line using Elph software [35].

Estimation the Rate of Synaptic Vesicle Exocytosis at Intense Activity

The rate of SV exocytosis was assessed using FM1-43 dye, as described previously in details [33, 36]. Briefly, FM1-43 dye (Cat. # T-35356, Thermo Fisher Scientific) reversibly binds to surface membranes and is then taken up by endocytosis into SVs. Next, nerve stimulation causes exocytotic release of FM1-43 from SVs [37]. To load FM1-43 into SVs, phrenic nerve was stimulated at 20-Hz for 1 min, thereby triggering massive SV exocytosis following endocytosis. FM1-43 (7 μM) was present in the external solution ½ min before 20 Hz stimulation, during stimulation and 5 min after the end of stimulation. Next, the muscles were washed for 20 min with dye-free physiological saline containing 20 μM ADVASEP-7 (Cat. # 70,029; Biotium). The latter facilitates the dissociation of FM1-43 from the surface membrane. Dye-loaded nerve terminals were treated with chemicals and re-stimulated at 20 Hz for 10 min to provoke SV exocytosis, leading to FM1-43 release from SVs (“unloading” or “destaining”). Fluorescence of FM1-43 was excited by a 480/10 nm light, and emission was recorded using a 535/40 nm filter.

FM1-43 fluorescence (∆F = FROI-Fback) was calculated in nerve terminals as a mean pixel intensity (in arbitrary units, a.u.) after background signal subtraction (Fback), which was determined in a region (4 × 30 μm2) outside the nerve terminals. A value of nerve terminal fluorescence (immediately before unloading stimulation; ∆Fmax) was set as 1.0 and a ratio of ∆F/∆Fmax was calculated during the unloading stimulation at 20 Hz. ∆F/∆Fmax did not change under resting conditions for 10 min (without nerve stimulation).

Fluorescence was detected using high-sensitive cameras, Dhyana 400BSI V2 (Tucsen) or DP72 (Olympus), and BX51WI microscope equipped by confocal disk scanning unit (Olympus) and CoolLED pE-4000 light source (CoolLED Ltd.). The cameras were controlled by Mosaic (Tucsen) and Cell P (Olympus) software. The regions containing NMJs were illuminated only at the moments of image recording ( < 1 second). Image Pro software (Media Cybernetics) was used to analyze fluorescence intensity.

Intracellular ROS Imaging

Detection of cytosolic ROS in NMJs was carried out using CM–H2DCFDA (#C6827; ThermoFisher Scientific) as described previously [34]. CM–H2DCFDA permeates the cells, and becomes trapped in the cytoplasm after hydrolysis by intracellular esterases. Muscles were exposed to 5 μM CM–H2DCFDA for 30 min, rinsed for 50 min with dye-free physiological solution and visualized with Olympus BX51 fluorescent microscope. CM–H2DCFDA fluorescence was evoked by a light of wavelength at 480/15 nM and the emission was recorded using a 505–550 nm band-pass filter. Excessive illumination was strictly avoided and preparations characterized by unstable fluorescence under basal conditions were excluded. Sample illumination was limited to 9 frames at 5 min intervals for 40 min to minimize photo-oxidation of the dye. This protocol allows to overcome the limitations of the dye [38, 39]. The intensity of fluorescence was quantified using Image Pro software and relative changes in fluorescence from baseline were estimated as previously described [34].

Measurements of Extracellular H2O2

The extracellular H2O2 levels were estimated with Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Cat. # A22188; ThermoFisher Scientific) as described previously [32, 34]. Briefly, in the presence of horseradish peroxidase Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) reacts with H2O2 (stoichiometry of 1:1) to produce resorufin, the stable red-fluorescent product. Basal levels of H2O2 were estimated after exposure of the preparation for 15 min to physiological saline (total volume of 400 μl) containing 100 μM Amplex Red reagent and 0.2 U/ml horseradish peroxidase. The bathing solution was mixed constantly by a recirculating mechanism. Then, the bathing solution was collected, stored for 50 min in the dark at room temperature and fluorescence was recorded. The fluorescence of resorufin was excited by 535/10 nM and the emission was detected at 590 nM. Background fluorescence derived from non-H2O2 reactions was subtracted from each value. For calibration a set of physiological solution with different H2O2 concentrations was used.

Assessment of Cytosolic Ca2+ Changes

Estimation of cytosolic Ca2+ changes in synaptic regions was performed using Oregon Green 488 BAPTA-1 (Cat. # O6807, Thermo Fisher Scientific) as described previously [32]. This is a cell-permeable high-affinity Ca2+ indicator (Kd ~ 170 nM) which allows to record small changes in cytosolic Ca2+ content near the resting levels. The preparations were incubated for 20 min with 2 μM Oregon Green 488 BAPTA-1 and then rinsed for 40 min with dye-free physiological solution. Finally, recording of fluorescence in synaptic regions were performed. Fluorescence was evoked by 1-s light flashes at 488/10 nm and the emission was detected with a 505–550 nm band‐pass filter. The value of fluorescence before chemical application was set to 1.0.

Statistics

OriginPro software was used for statistical analysis. Data are represented as mean ± standard deviation (SD); all exceptions are indicated (Fig. 3A, B). Sample size (n) is a number of independent experiments on individual animals and it was chosen based on a reasonable value of SD; sample size is denoted on each figure legend. Normality and variance homogeneity were tested by Shapiro–Wilk test and two-sample F test for variance, respectively. Statistical significance was assessed by a two-tailed paired t-test (for parametric and paired data) and Mann–Whitney U test (for comparison of two unpaired groups). Values of *P < 0.05, **P < 0.01, ***P < 0,001 were considered as significant.

Results

P2Y13 Receptors are Involved in Regulation of Spontaneous Exocytosis and Evoked Neurotransmitter Release at Low Frequency Stimulation

Previously, it was found that Gi protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release belong to P2Y13 subtype at the mouse motor nerve terminals [11, 40]. Rodent P2Y13 receptors are activated by both ADP and IDP [41, 42]. Expectedly, ADP (10 μM) and IDP (100 μM) reversibly reduced the amplitude of EPCs elicited by low-frequency (0.05 Hz) stimulation of the phrenic nerve (Fig. 1A, B).

Fig. 1
figure 1

Influence of ADP and IDP on evoked and spontaneous neurotransmitter release. A Representative multiquantal EPCs before and 15 min after addition of 10 μM ATP or 100 μM IDP in control and after treatment with 10 μM MRS 2211 (MRS). B Histograms showing the action of ADP (n = 11 mice) and IDP (n = 8) in control, and in the presence of MRS 2211 (n = 6). C–D Rise and decay time of EPCs before and after treatment with ADP (C) and IDP (D). E–G Graphs show amplitude values and the representative traces of MEPCs (E), rise and decay time (F) as well as frequency of MEPCs (G) before and after treatment with 10 μM ADP. n = 15. B–G: **P < 0.01, ***P < 0.001—by a Mann–Whitney U test between groups; ###P < 0.001 by two-tailed t-test as compared to pretreatment baseline

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By 15th min of ADP or IDP administration the EPC amplitude decreased by 29 ± 8% (n = 11, p = 0.003) or 37 ± 9% (n = 8, p = 0.007), respectively (Fig. 1 A, B). Under these conditions, the EPC kinetics (assessed by rise and decay time) was not affected (Fig. 1C, D). MRS 2211 (10 μM), a selective P2Y13 receptor antagonist, completely prevented the inhibitory effect on EPC amplitude of both ADP and IDP (Fig. 1A, B). Importantly, that MEPC amplitude as well as rise and decay time, indicators of postsynaptic membrane sensitivity to ACh, were not changed upon ADP application (Fig. 1E, F). At the same time, MEPC frequency decreased from 1.0 ± 0.2 to 0.6 ± 0.1 s−1 (p < 0,001; n = 15) by 15th min of ADP administration (Fig. 1 G). Thus, ADP via activation of P2Y13-receptors suppressed both spontaneous exocytosis and evoked neurotransmitter release upon single stimuli in the mouse NMJs.

Purinoceptor-Dependent Modulation of the Rate of Synaptic Vesicle Exocytosis During Intense Activity

During quiet breathing phrenic motor neurons discharge at 20–30 Hz [43]. This stimulation mobilizes SVs from different pools in the NMJs [44, 45]. Moreover, the efficiency of neurotransmission at intense activity depends mainly on SV delivery to active zone and their recycling [46, 47]. In this section, using FM1-43 dye, we tested the contribution of purinergic receptors in regulation of SV recruitment into exocytosis during prolonged nerve stimulation at 20 Hz (Fig. 2).

Fig. 2
figure 2

Purinergic regulation of synaptic vesicle recruitment to exocytosis during phrenic nerve stimulation at 20 Hz. A, B The kinetics of FM1-43 dye unloading from motor nerve terminals in control (Cntr), after treatment with ADP, P2Y13-receptor antagonist MRS 2211 (MRS), and ADP in the presence of MRS 2211. n = 11 (Cntr), 11 (ADP), 7 (MRS) and 10 (MRS + ADP). C Representative fluorescent images immediately before (0) and at different time points (180 and 600 s) of unloading stimulation. Scale bars—15 µm. D Quantification of dye loss portions by 60 and 600 s of stimulation; data calculated from A and B. **P < 0.01, ***P < 0.001 by a Mann–Whitney U test between groups; ns non significant. A, B and D: data are represented as mean ± SD

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Motor nerve terminals were loaded with fluorescent FM1-43 dye by 20 Hz stimulation for one minute. Then, preloaded nerve terminals were re-stimulated at 20 Hz to induce the exocytotic dye release (unloading) in control and after treatment with chemicals (Fig. 2A–C). ADP (10 μM) decreased the rate of FM1-43 unloading during 20 Hz activity (Fig. 2A). As a result, 27 ± 20% (p = 0.006, n = 11) and 31 ± 12% (p < 0.001) less dye was released by 60 and 600 s of the stimulation, respectively, as compared to control (Fig. 2D). A competitive antagonist of P2Y13 receptors, MRS 2211 (10 μM), enhanced the rate of FM1-43 unloading (Fig. 2B), causing an increase in FM1-43 dye portion release by 54 ± 21% (p < 0.001, n = 7) and 15 ± 5% (p = 0.003) after 60 and 600 s of the stimulation, respectively (Fig. 2D). In the presence of MRS 2211, the suppressive effect of ADP on exocytotic dye release was markedly reduced (Fig. 2B) and the dye loss was only by 15 ± 17% (p = 0.053, n = 10) less after 600 s of stimulation as compared to control (Fig. 2D).

Thus, exogenous ADP acting mainly via P2Y13 receptors suppressed SV involvement in exocytosis during prolonged 20 Hz activity. Activation of P2Y13 receptor by endogenous ATP released at intense activity can contribute to negative regulation of the rate of SV exocytosis.

The Contribution of Purinoceptors to ROS Production: Signaling Pathways

Assessment of Cytoplasmic ROS Levels

ROS imaging using the CM-H2DCFDA dye, which upon oxidation by intracellular ROS forms highly fluorescent carboxy-dichlorofluorescein, provided further insights into the mechanism of ATP/ADP action. 15-min application of 10 μM ADP markedly increased CM-H2DCFDA fluorescence by 177 ± 88% (n = 13, p < 0.001) in the synaptic regions. (Fig. 3A, B). This enhancement of cytosolic ROS began with a 5-min lag period after the addition of ADP. Hypothetically, lipid rafts may be involved in ADP-mediated elevation of ROS. Previously, we found a functional coupling of P2Y12-receptor/NOX axis to lipid rafts at the frog NMJs [9]. Sphingomyelin-hydrolyzing enzyme SMase at low concentration (0.01 U/ml) can disrupt the lipid rafts mainly in synaptic zones of mouse NMJs [30]. SMase preapplication completely eliminated the ADP effect on CM-H2DCFDA fluorescence (Fig. 3B). An increase in CM-H2DCFDA fluorescence was also observed during 20 Hz stimulation of the phrenic nerve. By 1st, 5th and 10th min of the stimulation, the fluorescence intensity was 116 ± 18% (n = 10, p = 0.01), 164 ± 39% (p < 0.001) и 222 ± 68% (p < 0.001) of the pre-stimulation value, respectively (Fig. 3 C, D). This may be explained by the effect of sufficient amounts of endogenous ATP released from nerve terminals during intense activity [48]. CM-H2DCFDA allows to detect the time course of ROS production but has certain limitations [38].

Fig. 3
figure 3

ADP increases production of ROS. A Time course of ADP action on fluorescence of ROS-sensitive dye (CM-H2DCFDA) at junctional regions in control and after SMase preexposure. B Quantification of changes in CM-H2DCFDA fluorescence after 15 min exposure to ADP in control (n = 13) and SMase-pretreated samples (n = 8). Right, typical fluorescence images in control and after treatment with ADP alone and SMase + ADP. A, B The values immediately before ADP administration were set as 100%. C Time course of CM-H2DCFDA fluorescence in junctional regions before (0–15 min) and during (15–25 min) stimulation (10 min, 20 Hz) of phrenic nerve. Inserts, the representative fluorescent images captured at different time point before (15 min) and after (25 min) the stimulation. D Quantification of changes in CM-H2DCFDA fluorescence by 1st (16 min), 5th (20 min) and 10th min (25 min) of stimulation. C, D Values immediately before onset of the stimulation were set as 100%. E Action of ADP on levels of extracellular H2O2, detected by Amplex Red-based assay (n = 7). Control (cntr) indicates values before ADP application (in a.u.). The fluorescence was compared to H2O2 calibration curve to evaluate the levels of H2O2 (right Y axis). F Quantification of relative changes in resorufin fluorescence after ADP application in control (n = 7), after pretreatment with VAS 2870 (n = 5) and applying PTX (n = 6). Also, the effects of phrenic nerve stimulation (20 Hz, 15 min, n = 4) or high K+-induced membrane depolarization (40 mM K+, 15 min, n = 6) itself and in combination with PTX (n = 5) or MRS 2211 (n = 6) treatment are shown. B, C: Scale bars – 15 μm. A, C and B, D, E, F data are represented as mean ± SE and ± S.D, respectively. #P < 0.05, ##P < 0.01, ###P < 0.001 by paired two-tailed t-test as compared to baseline; **P < 0.01, ***P < 0.001—by a Mann–Whitney U test between groups

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The main signaling form of ROS is H2O2, which has a relatively long half-life in living cells and can cross cell membranes [49,50,51]. Therefore, a specific sensor for extracellular H2O2 was used in further experiments.

Evaluation of Extracellular Levels of H2O2

The levels of extracellular H2O2 were estimated using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit [9, 34]. 15 min application of ADP (10 μM) increased the extracellular content of H2O2. The fluorescence of resorufin, the product of Amplex Red reagent oxidation by H2O2, was elevated by 40 ± 36% (n = 7; p < 0.002) after ADP treatment (Fig. 3E).

Plasmalemmal NOX, a possible source of ROS, produces mainly extracellular superoxide radical anion, which is dismutated into H2O2 [52]. ADP-induced enhancement of resorufin fluorescence was prevented by VAS 2870 (10 µM), a selective inhibitor of NOX, as well as inhibition of Gi protein with PTX (150 μg/kg) (Fig. 3F). Hence, ADP, acting on Gi protein-coupled P2Y receptors, increased H2O2 production involving NOX.

To test production of extracellular H2O2 during neuromuscular transmission, massive SV exocytosis was induced by either motor nerve stimulation (20 Hz) or membrane depolarization (by high K+-containing saline) for 15 min. In both cases, resorufin fluorescence increased by 93 ± 12% (n = 4) and 27 ± 3% (n = 6, p < 0.001), respectively (Fig. 3F). These data are consistent with the assessment of the increase in intracellular ROS levels in response to motor nerve stimulation (Fig. 3C, D). As with the ADP effect, the nerve stimulation-induced enhancement of H2O2 levels was precluded by Gi protein blockage (Fig. 3F). Importantly, that P2Y13 antagonist MRS 2211 profoundly suppressed the depolarization-induced elevation of resorufin fluorescence (Fig. 3F) and under these conditions the fluorescence increased only by 11 ± 1% (n = 6; p = 0.004).

Taken together, these data (3.3.1 and 3.3.2 sections) suggest that purinoreceptor activation with ADP enhances ROS production in a lipid raft, Gi protein and NOX-dependent manner. Intense synaptic activity also can increase ROS generation, at least in part, via activation of P2Y13 receptors and Gi protein.

Contribution of ROS-Related Signaling in Purinergic Regulation of the Evoked Neurotransmitter Release at Low Frequency Stimulation

Next, we tested the action of ADP in the presence of several antioxidants known to prevent the cellular effects of ROS. First, we used 1 mM N-acetylcysteine (NAC), a membrane-permeable antioxidant, which itself increased the EPC amplitude to 136 ± 14% (n = 5). Pretreatment with NAC significantly reduced the negative action of 10 μM ADP (Fig. 4). By 15th min of ADP application, the EPC amplitude decreased only by 10 ± 8% (p < 0.001, n = 9), while in control the decline of EPC amplitude was approximately 3 times more pronounced (p < 0.001, n = 11). To explore the nature of the ROS which could be involved in the action of ADP, catalase, a non-membrane permeable enzyme which specifically destroys H2O2, was applied. In our experiments 1200 U/ml catalase increased the amplitude of EPCs by 14 ± 5% (p < 0.05, n = 6) and significantly reduced the inhibitory action of 10 μM ADP (Fig. 4). In the latter case, ADP decreased the EPC amplitude by 7 ± 7% (p < 0.001; n = 10). These results suggest the involvement of ROS, specifically extracellular H2O2, in the action of ADP on evoked neurotransmitter release.

Fig. 4
figure 4

Testing the role of ROS, NOX, PKC and lipid microenviroment in the action of ADP. Histogram shows effects (in %) of ADP on amplitude of EPCs in control and after pre-exposure to N-acetylcysteine (NAC) (n = 9), catalase (n = 10), VAS2870 (n = 6), chelerythrine (Chele) (n = 7) and 0.1 U/ml SMase (n = 6). **P < 0.01, ***P < 0.001—by a Mann–Whitney U test between groups. ##P < 0.01, ###P < 0.001 by paired two-tailed t-test as compared to value before ADP addition

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Probably, the main source of ROS associated with the synaptic action of ADP could be NOXs. Indeed, pretreatment with NOX inhibitor VAS2870 significantly reduced the negative action of 10 μM ADP and 15 min after ADP addition the EPC amplitude decreased only by 11 ± 2% (Fig. 4). Molecular link between Gi protein coupled P2Y13 receptor and activation of NOX can be protein kinase C (PKC) [53]. The latter may be activated via Gi protein/Gβγ/phospholipase C pathway [32, 54, 55]. Cell-permeable protein kinase C inhibitor chelerythrine chloride (Chele), completely suppressed the effect of ADP on the EPC amplitude (Fig. 4). Note that ADP-dependent ROS generation was prevented by lipid raft-disrupting manipulation, particularly SMase pretreatment (Fig. 2A, B). Similarly, SMase markedly attenuated ADP-induced suppression of the EPC amplitude (Fig. 4).

Involvement of PKC and NADPH Oxidase in Purinergic Regulation of the Exocytotic Rate at Intense Activity

Here we tested the involvement of NOX and PKC in the ADP-mediated suppression of SV exocytosis during intense activity (20 Hz, 10 min). Inhibitor of NOX itself decreased the rate of FM1-43 unloading vs control (Fig. 5A, C). After inhibition of NOX, ADP lost its ability to decrease the exocytotic FM1-43 release and, conversely, increased the rate of FM1-43 destaining as compared to VAS2870-treated NMJs (Fig. 5A, C). As a result, 43 ± 14% (n = 8, p < 0.001) more dye was released for 600 s of 20 Hz stimulation in VAS + ADP vs VAS-treated NMJs (Fig. 5C). Inhibition of PKC, which can be responsible for NOX activation, did not modify FM1-43 dye unloading (Fig. 5B), but completely prevented ADP-mediated changes in the rate of FM1-43 release and, hence, amount of dye loss during 20 Hz stimulus train (Fig. 5B, C). Hence, ADP-induced activation of NOX in a PKC-dependent manner can mediate the negative effect of P2Y13 receptors on SV recruitment to exocytosis at intense activity.

Fig. 5
figure 5

Involvement of NADPH oxidase and protein kinase C in the effect of ADP on the rate of exocytotic FM-dye release during prolonged 20 Hz stimulation of phrenic nerve. A, B The kinetics of FM1-43 dye release from nerve terminals during stimulation after inhibition of NOX (A) or PKC (B) with VAS2870 (VAS) or chelerythrine (Chele), respectively. n = 12 (VAS), 8 (VAS + ADP), 8 (Chele) and 12 (Chele + ADP). Control curve is also shown on A and B (from Fig. 2A). C Quantification of FM1-43 dye loss for 60 and 600 s of the unloading stimulation under conditions of NOX or PKC blockage. n = 8–12 per group. *P < 0.05, ***P < 0.001—by a Mann–Whitney U test between groups; ns Non significant

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Purinergic Regulation of Cytosolic Ca2+

PKC-dependent NOX activation can require calcium release from intracellular store [56]. We hypothesize that ADP, via P2Y13-receptors, may cause a rapid elevation of cytosolic Ca2+ before activation of NOX causing ROS generation, which begins with a 5-min delay after the addition of ADP. Indeed, application of ADP led to a rapid increase in intracellular Ca2+ levels in synaptic regions (Fig. 6A, D). The fluorescence of Oregon Green® 488 BAPTA-1 increased by 27 ± 4% (n = 8, p = 0.001) within the first 3 min of ADP administration. This rise in the fluorescence was completely prevented by both P2Y13 receptor antagonist MRS 2211 (10 µM) (Fig. 6B, D) and inhibitor of Ca2+ release from endoplasmic reticulum TMB8 (10 μM) (Fig. 6C, D). Furthermore, TMB8 completely prevented the ADP-mediated suppression of exocytotic FM1-43 dye release upon prolonged 20 Hz stimulation (Fig. 6E, F). TMB8 itself slightly attenuated FM1-43 unloading and 16 ± 12% less dye (n = 7, p = 0.014) was released after 600 s of 20 Hz stimulation as compared to control (Fig. 6F). Thus, the elevation of cytosolic Ca2+ due to its release from intracellular store is an early event in P2Y13 receptor-dependent signaling and required for ADP-induced suppression of SV involvement into exocytosis.

Fig. 6
figure 6

P2Y13 receptor-dependent changes in cytosolic calcium in synaptic regions. A–C Time course of Oregon Green 488 BAPTA-1 fluorescence (Ca2+ indicator) in synaptic regions in control (Cntr), after addition of ADP, MRS 2211 (MRS), TMB8 and ADP in combination with MRS 2211 or TMB8. A: insert, typical fluorescent images before (0) and 180 s after ADP/vehicle application; scale bars—15 µm. n = 7 (Cntr), 8 (ADP), 10 (MRS), 10 (MRS + ADP), 8 (TMB) and 8 (TMB + ADP). D Quantification of fluorescence changes by 180 s of the chemical application. E FM1-43 unloading curves in TMB and TMB + ADP-treated NMJs. n = 7 per group. F Quantification of FM1-43 portion loss after 60 and 600 s of stimulation at 20 Hz. D, F: *P < 0.05, **P < 0.01, ***P < 0.001—by a Mann–Whitney U test between groups; ns Non significant. A–F: data are represented as mean ± SD

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The Role of Lipid Raft Component in Purinergic Regulation of Synaptic Vesicle Involvement to Exocytosis

Sphingomyelin and cholesterol are building blocks for plasmalemmal microdomains, lipid rafts [57]. Enzymatic sphingomyelin hydrolysis (by SMase) and cholesterol oxidation (by ChO) were used to modulate the membrane properties, interfering with lipid raft signaling [58,59,60]. In addition, SMase treatment causes accumulation of ceramide, an active lipid and a player in neurological pathologies, at the plasmalemma [61]. Previously, we demonstrated that SMase (0.01 u/ml) and ChO (0.2 u/ml) partially disrupt lipid raft phase mainly in NMJ regions and enhance SV mobilization [30, 31]. Consistent with these data, SMase and ChO increased the rate of FM1-43 unloading (Fig. 7). After SMase pretreatment, the suppressive effect of ADP on FM1-43 unloading was attenuated (Fig. 7A, D), whereas action of P2Y13 receptor inhibitor was even inverted (Fig. 7B vs Fig. 2B), and MRS 2211 suppressed exocytotic FM-dye release during 20 Hz nerve stimulation (Fig. 7B). Indeed, 21 ± 6% less (n = 7, p = 0.001) FM1-43 dye was released by 600 s of 20 Hz stimulation in SMase + MRS 2211 vs SMase-treated NMJs (Fig. 7D). Similarly, following ChO pretreatment MRS 2211 lost its ability to increase the rate of FM1-43 unloading (Fig. 7C, D).

Fig. 7
figure 7

The role of lipid microenvironment in purinergic regulation of exocytotic release at 20 Hz activity. A–C influence of ADP (A) and P2Y13 antagonist MRS 2211 (B, C) on the kinetics of FM1-43 unloading after pretreatment with sphingomyelinase (A, B) or cholesterol oxidase (C). The unloading was induced by phrenic nerve stimulation. Control (Cntr), ADP and MRS curves from Fig. 2A, B are also shown. n = 9 (SMase), 12 (SMase + ADP), 8 (SMase + MRS), 12 (ChO) and 8 (ChO + MRS). D Quantification of FM1-43 dye portion released for 60 and 600 s of stimulation at 20 Hz. **P < 0.05, ***P < 0.001—by a Mann–Whitney U test between groups; ns non significant. A–D: data are represented as mean ± SD

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Discussion

A large body of literature shows that extracellular nucleotides play an important role in intercellular signaling engaging inotropic (P2X) and metabotropic (P2Y) receptors in the nervous system [7, 8, 62]. Among metabotropic receptors, P2Y13 subtype is one of the most recently cloned [63] and, together with the P2Y1 and P2Y12 subtypes, P2Y13 receptors belong to a distinct structural branch of the P2Y receptors, which are more specific for ADP vs ATP [64]. In addition, P2Y13 as well as P2Y12, and P2Y14 receptors are coupled preferentially to Gi protein [63, 65].

P2Y13 receptors seem to play an essential role in nervous system. Investigation of P2Y13 receptor distribution in rat tissues demonstrated the highest expression levels in spleen, followed by liver and brain with particularly high levels in cortex and striatum [66]. Furthermore, P2Y13 receptors were revealed in rat spinal cord, dorsal root ganglion [67], superior cervical ganglion [68] and mouse motor nerve terminals [11]. In latter case, Gi protein-coupled P2Y13 receptors are considered as the best candidates for ATP/ADP-dependent negative feedback control of ACh release in the mammalian NMJs [11, 17].

ATP is one of the most common co-transmitters in both central and peripheral nervous system. In the synaptic cleft it is enzymatically hydrolyzed to ADP, AMP and finally adenosine [7, 8]. In addition, contracting muscle fibers [69, 70] and Schwann cells [71, 72] could be sources of extracellular purines. Both extracellular adenosine and ATP/ADP mainly reduce neurotransmitter release via binding to adenosine-and P2Y-receptors in the amphibian and mammalian NMJs [11, 16, 40, 73,74,75,76].

Consistent with previous studies [11, 17], we showed that ADP and IDP reduce spontaneous exocytosis and evoked neurotransmitter release in response to low frequency stimulation in the mouse NMJs. In addition, experiments with tracking of exocytotic FM dye release demonstrated that ADP decreases the rate of exocytosis during intense activity. A selective antagonist of P2Y13 receptors suppressed these effects of purinergic agonists, thereby pointing to P2Y13 receptors as a main target for extracellular purines in the mouse NMJs. Importantly, the amplitude and kinetics of MEPCs were not affected, suggesting a pure presynaptic action of purinergic agonists.

The levels of endogenous purines at low frequency activity are not enough to regulate neurotransmitter release. Only induction of massive SV exocytosis seems to cause an elevation of endogenous purines concentration above purinergic receptor activation threshold [48]. Indeed, inhibitor of P2Y13 receptors enhanced the rate of FM1-43 dye release during nerve stimulation at 20 Hz, a more physiological mode of activity for phrenic motoneurons [43]. This points to suppression of SV involvement in exocytosis at intense activity due to activation of P2Y13 receptors by endogenous purines.

ROS as Second Messengers in ADP Action in the NMJs

Activation of P2Y receptors can lead to production of ROS as well as reactive nitrogen species, which affect multiple redox-dependent processes in cells [77, 78]. In the frog NMJs, ROS play a key role in P2Y12 receptor-dependent action of ATP on neurotransmitter release [9, 13].

Previously, it was shown that phrenic nerve stimulation significantly increases extracellular concentrations of ATP [69, 79], which can activate P2Y13 receptors at the mouse NMJs [48]. Here we demonstrated that both exogenous ADP and phrenic nerve activation increase intracellular ROS to comparable levels. In addition, estimation of extracellular levels of H2O2, the main signaling form of ROS [49,50,51], revealed an elevation of H2O2 generation upon both ADP application and phrenic nerve stimulation. This increase in H2O2 production was precluded by PTX, suggesting an involvement of Gi protein-coupled P2Y13 receptors [11, 65].

High K+ induces depolarization of the nerve terminals, causing quantal release of ACh and ATP [17, 80]. As a result, ATP/ADP and adenosine in the synaptic cleft can activate different purinergic receptors, particularly P2Y13 and A1, A2A and A3 subtypes [17]. We found that, similar to nerve stimulation, high K+-induced membrane depolarization increases external H2O2 levels. This enhancement of H2O2 generation was markedly attenuated by P2Y13 receptor antagonist. It is important to emphasize that the detected changes in H2O2 concentration correlate well with the physiological range of hydrogen peroxide amounts [81].

Main sources of O2− and H2O2 in cells are transmembrane NOXs and electron transport chain of mitochondria [82, 83]. NOXs also can be associated with specialized endosomes (redoxosomes), this allows to compartmentalize H2O2-mediated signaling [84]. We found that ADP-induced H2O2 production in the neuromuscular preparations was completely blocked by VAS 2870, a specific NOX blocker [85]. In addition, VAS 2870 markedly attenuated ADP-mediated reduction of evoked ACh release in response to single stimuli and prevented ADP-induced suppression of exocytic FM1-43 dye loss during intense activity. These data suggest a key role of NOX-derived ROS in P2Y13 receptor-dependent regulation of neuromuscular transmission. Indeed, antioxidants, NAC [86] and catalase [87], decreased the effect of ADP on neurotransmitter release. Note that catalase does not penetrate into cells, hence it should mainly neutralize extracellular H2O2, which is produced by plasma membrane bound NOXs [88].

In microglia and astrocytes, activation of P2Y12 and P2Y13 receptors caused Ca2+ mobilization [64, 89]. In mammalian NMJs, Gi protein-dependent Ca2+ release from intracellular store can increase H2O2 production [32]. We also found that ADP causes an increase in cytosolic Ca2+ (with the peak within 2–3 min) which is prevented by both P2Y13 receptor antagonist (MRS 2211) and blocker of Ca2+ mobilization from endoplasmic reticulum (TMB8). Importantly, the elevation of [Ca2+]in occurred before increase in intracellular ROS levels. Like NOX antagonist, inhibition of Ca2+ mobilization precluded suppressive action of ADP on the rate of exocytosis during intense activity. Accordingly, P2Y13 receptor-dependent Ca2+mobilization can contribute to NOX activation and suppression of exocytosis.

Increase in cytosolic Ca2+ can promote activation of NOX via Ca2+-dependent signaling pathways engaging multiple players, e.g. PKC, MAP kinase or phospholipase A2 [90]. P2Y receptor-mediated Ca2+ mobilization is able to activate NOX2 through PKC in the immune cells [91]. We found that blocker of PKC, chelerythrine, acting in a similar manner as antioxidants and NOX inhibitor, suppresses ADP-induced decrease in evoked neurotransmitter release at low frequency stimulation. Furthermore, chelerythrine precluded ADP-induced changes in the rate of SV exocytosis at intense activity. Hence, P2Y13 receptor-dependent attenuation of synaptic transmission can rely on Gi protein/Ca2+/PKC/NOX/ROS signaling in the mouse NMJs.

G protein-coupled receptors as well as signaling molecules, including NOXs, can reside in the cholesterol and sphingomyelin-rich microdomains [9, 92, 93], which are likely to be abundant at the NMJs [22, 24, 94]. Previously, we found that exposure to SMase or ChO causes a partial lipid raft disruption and it increases the rate of SV exocytosis during intense activity at the diaphragm NMJs [30, 31]. Furthermore, the effect of SMase on neurotransmission was sensitive to Gi protein inhibition [30]. This suggests that lipid raft disruption might enhance neurotransmitter release by interfering with signaling via Gi protein-coupled receptors. Particularly, one of the reasons for the enhancement of neurotransmission can be attenuation of P2Y13 receptor-dependent negative feedback regulation of exocytosis. Consistent with this hypothesis is that after ChO and SMase pretreatment blockage of P2Y13 receptors lost its ability to enhance the rate of SV exocytosis at intense activity. In addition, SMase pretreatment inhibited the effects of exogenous ADP on evoked SV exocytosis and intracellular ROS levels. Hence, lipid-raft disrupting manipulations blunted ADP/P2Y13 receptor-mediated suppression of neurotransmission in the mouse NMJs. Similarly, cholesterol depletion reduced and largely delayed the action of ATP via P2Y12 receptors at the frog NMJs [9].

Interestingly, after exposure to SMase inhibition of P2Y13 receptors caused the opposite effect, i.e. a decrease in the exocytotic rate at intense activity. Speculatively, changes in lipid microenvironment and (or) ceramide accumulation after sphingomyelin hydrolysis can lead to a switching of P2Y13 receptor-dependent signaling to Gs protein. The latter can trigger cascades causing an increase in synaptic vesicle exocytosis [95, 96]. Although P2Y13 receptors are mainly coupled to Gi protein, under some conditions, they can trigger signaling via Gs proteins [63, 97, 98]. Recently, we found that plasmalemmal sphingomyelin hydrolysis inverses the effects of β2-adrenergic agonist on neuromuscular transmission via switching to Gi protein-dependent signaling [99]. Thus, destabilization of lipid rafts and sphingomyelin hydrolysis can strongly interfere with both para-and endocrinal mechanisms of neurotransmission control, thereby contributing to the progression of neuromuscular disorders.

Lipid raft disruption in the NMJs can occur at the early stages in amyotrophic lateral sclerosis [27, 28] and during short-term muscle disuse [25], whereas overexpression of a lipid raft organizer protein caveolin had beneficial actions in models of amyotrophic lateral sclerosis [100, 101]. Furthermore, upregulation of SMase was demonstrated in conditions associated with skeletal muscle atrophy and damage [102,103,104,105,106]. According to this scenario, loss of the negative feedback control mechanism can lead to excessive neurotransmission and ACh release. Higher levels of ACh at the synaptic cleft may promote the degeneration of adult NMJs, contributing to disease-related deteriorations of motor function [6]. By contrast, increased neurotransmitter release can also be a compensatory response to keep the safety factor of neuromuscular transmission at the required level under pathological conditions or during aging [107]. In addition, activity of nicotinic ACh receptors can counteract muscle fiber atrophy and favor reinnervation [108].

Conclusions

Co-transmitter ATP and its derivatives mediate a negative feedback loop that limits excessive ACh release in the mammalian NMJs. Our data indicates that ATP/ADP, via activation of the P2Y13 receptor, triggers sequential Ca2+ mobilization and NOX-dependent ROS production. Generated ROS (particularly H2O2) attenuate SV exocytosis induced by both low and higher frequency stimulation of the phrenic nerve. Targeting key components of lipid rafts suppresses P2Y13 receptor-mediated effects on both exocytosis and ROS levels, pointing to the key meaning of lipid microenvironment for purinergic modulation of neuromuscular transmission.