Quantal size increase induced by the endocannabinoid 2-arachidonoylglycerol requires activation of CGRP receptors in mouse motor synapses.

In mouse motor synapses, the exogenous application of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG) increases acetylcholine (ACh) quantal size due to the activation of CB1 receptors and the stimulation of ACh vesicular uptake. In the present study, microelectrode recordings of miniature endplate potentials (MEPP) revealed that this effect of 2-AG is independent of brain-derived neurotrophic factor (BDNF) signaling but involves the activation of calcitonin gene-related peptide (CGRP) receptors along with CB1 receptors. Potentiation of MEPP amplitude in the presence of 2-AG was prevented by blockers of CGRP receptors and ryanodine receptors (RyR) and by inhibitors of phospholipase C (PLC) and Ca2+ /calmodulin-dependent protein kinase II (CaMKII). Therefore, we suggest a hypothetical chain of events, which starts from the activation of presynaptic CB1 receptors, involves PLC, RyR, and CaMKII, and results in CGRP release with the subsequent activation of presynaptic CGRP receptors. Activation of CGRP receptors is probably a part of a complex molecular cascade leading to the 2-AG-induced increase in ACh quantal size and MEPP amplitude. We propose that the same chain of events may also take place if 2-AG is endogenously produced in mouse motor synapses, because the increase in MEPP amplitude that follows after prolonged tetanic muscle contractions (30 Hz, 2 min) was prevented by the blocking of CB1 receptors. This work may help to unveil the previously unknown aspects of the functional interaction between ECs and peptide modulators aimed at the regulation of quantal size and synaptic transmission.

ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses.

The effects of brain-derived neurotrophic factor (BDNF) processing by-products (proBDNF and BDNF prodomain) on the activity of mouse neuromuscular junctions (NMJs) were studied in synapses formed during the reinnervation of extensor digitorum longus muscle (m. EDL) and mature synapses of the diaphragm. The parameters of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were analyzed in presence of each of the BDNF maturation products (both - 1 nM). In newly formed NMJs, proBDNF caused an increase in the resting membrane potential of muscle fibers and a decrease in the frequency of MEPPs, which was prevented by tertiapin-Q, a G-protein-coupled inwardly rectifying potassium channels (GIRK) blocker but not by p75 receptor signaling inhibitor TAT-Pep5. proBDNF had no effect on the parameters of EPPs. BDNF prodomain in newly formed synapses had effects different from those of proBDNF: it increased the amplitude of MEPPs, which was prevented by vesamicol, an inhibitor of vesicular acetylcholine (ACh) transporter; and reduced the quantal content of EPPs. In mature NMJs, proBDNF did not influence MEPPs parameters, but BDNF prodomain suppressed both spontaneous and evoked ACh release: decreased the frequency and amplitude of MEPPs, and the amplitude and quantal content of EPPs. This effect of the BDNF prodomain was prevented by blocking GIRK channels, by TAT-Pep5 or by Rho-associated protein kinase (ROCK) inhibitor Y-27632. At the same time, the BDNF prodomain did not show any inhibitory effects in diaphragm motor synapses of pannexin 1 knockout mice, which have impaired purinergic regulation of neuromuscular transmission. The data obtained suggest that there is a previously unknown mechanism for the acute suppression of spontaneous and evoked ACh release in mature motor synapses, which involves the activation of p75 receptors, ROCK and GIRK channels by BDNF prodomain and requires interaction with metabotropic purinoreceptors. In general, our results show that both the precursor of BDNF and the product of its maturation have predominantly inhibitory effects on spontaneous and evoked ACh release in newly formed or functionally mature neuromuscular junctions, which are mainly opposite to the effects of BDNF. The inhibitory influences of both proteins related to brain neurotrophin are mediated via GIRK channels of mouse NMJs.

Noncanonical Activity of Endocannabinoids and Their Receptors in Central and Peripheral Synapses.

This review focuses on new aspects of endocannabinoid functions and mechanisms of activity in central and peripheral synapses, different from the general viewpoint that endocannabinoids are retrograde signaling molecules, which inhibit neurotransmitter release by activating specific presynaptic endocannabinoid receptors CB1 and CB2. Biased agonism of the endogenous and synthetic cannabinoids as well as ability of the CB-receptors to couple not only with classical Gi-proteins, but also with Gs- and Gq-proteins and, moreover, with ß-arrestins (thereby triggering additional signaling pathways in synapses) are described here in detail. Examples of noncanonical tonic activity of endocannabinoids and their receptors and their role in synaptic function are also presented. The role of endocannabinoids in short-term and long-term potentiation of neurotransmitter release in central synapses and their facilitating effect on quantal size and other parameters of acetylcholine release in mammalian neuromuscular junctions are highlighted in this review. In conclusion, it is stated that the endocannabinoid system has a wider range of various multidirectional modulating effects (both potentiating and inhibiting) on neurotransmitter release than initially recognized. Re-evaluation of the functions of endocannabinoid system with consideration of its noncanonical features will lead to better understanding of its role in the normal and pathological functioning of the nervous system and other systems of the body, which has an enormous practical value.

Delayed increase of acetylcholine quantal size induced by the activity-dependent release of endogenous CGRP but not ATP in neuromuscular junctions.

In mouse motor synapses tetanic neuromuscular activity (30 Hz, 2 min) led to a delayed posttetanic potentiation of amplitude and duration of spontaneous miniature endplate potentials (MEPPs). Microelectrode recordings of MEPPs before and after nerve stimulation showed an increase in MEPP amplitude and time course by 30% and 15%, respectively, without changes in their frequency. Peak effect was detected 20 min after tetanic activity and progressively faded throughout the next 40 min of recording. The revealed potentiation of MEPPs was fully preserved in preparations from pannexin 1 knockout mice. It means, that myogenic ATP released via pannexin 1 channels from contracting muscle fibers is not likely to participate in the described phenomenon. But posttetanic potentiation of MEPPs was fully prevented by competitive antagonist of calcitonin gene-related peptide (CGRP) receptors CGRP8-37 , ryanodine receptors inhibitor ryanodine and by vesicular acetylcholine transporter inhibitor vesamicol. It is suggested that the combination of intensive synaptic and contractile activity in neuromuscular junctions is required to induce Ca2+ -dependent exocytosis of endogenous CGRP. The accumulation of CGRP in the synaptic cleft and its presynaptic activity may induce posttetanic potentiation of MEPP amplitude due to CGRP-stimulated acetylcholine loading into vesicles and subsequent increase of quantal size.

Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses.

The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1-/-) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 µM). In WT and Panx1-/- mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1-/- mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 µM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1-/- mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1-/- NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 µM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1-/- mice to above the control level.

Regulation of Acetylcholine Quantal Release by Coupled Thrombin/BDNF Signaling in Mouse Motor Synapses.

The aim of this study was to compare the acute effects of thrombin and brain-derived neurotrophic factor (BDNF) on spontaneous miniature endplate potentials (MEPPs) and multiquantal evoked endplate potentials (EPPs) in mouse neuromuscular junctions (NMJs) of m. diaphragma and m. EDL. Intracellular microelectrode recordings of MEPPs and EPPs were used to evaluate the changes in acetylcholine (ACh) release in mature and newly-formed mouse NMJs. Thrombin (1 nM) increased the amplitude of MEPPs and EPPs by 25-30% in mature and newly-formed NMJs. This effect was due to an enhanced loading of synaptic vesicles with ACh and increase of ACh quantal size, since it was fully prevented by blocking of vesicular ACh transporter. It was also prevented by tropomyosin-related kinase B (TrkB) receptors inhibitor ANA12. Exogenous BDNF (1 nM) mimicked thrombin effect and increased the amplitude of MEPPs and EPPs by 25-30%. It required involvement of protein kinase A (PKA) and mitogen-activated protein kinase (MEK1/2)-mediated pathway, but not phospholipase C (PLC). Blocking A2A adenosine receptors by ZM241385 abolished the effect of BDNF, whereas additional stimulation of A2A receptors by CGS21680 increased MEPP amplitudes, which was prevented by MEK1/2 inhibitor U0126. At mature NMJs, BDNF enhanced MEPPs frequency by 30-40%. This effect was selectively prevented by inhibition of PLC, but not PKA or MEK1/2. It is suggested that interrelated effects of thrombin/BDNF in mature and newly-formed NMJs are realized via enhancement of vesicular ACh transport and quantal size increase. BDNF-induced potentiation of synaptic transmission involves the functional coupling between A2A receptor-dependent active PKA and neurotrophin-triggered MAPK pathway, as well as PLC-dependent increase in frequency of MEPPs.

Mechanism of P2X7 receptor-dependent enhancement of neuromuscular transmission in pannexin 1 knockout mice.

P2X7 receptors are present in presynaptic membranes of motor synapses, but their regulatory role in modulation of neurotransmitter release remains poorly understood. P2X7 receptors may interact with pannexin 1 channels to form a purinergic signaling unit. The potential mechanism of P2X7 receptor-dependent modulation of acetylcholine (ACh) release was investigated by recording miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in neuromuscular junctions of wild-type (WT) and pannexin 1 knockout (Panx1-/-) mice. Modulation of P2X7 receptors with the selective inhibitor A740003 or the selective agonist BzATP did not alter the parameters of either spontaneous or evoked ACh release in WT mice. In Panx1-/- mice, BzATP-induced activation of P2X7 receptors resulted in a uniformly increased quantal content of EPPs during a short stimulation train. This effect was accompanied by an increase in the size of the readily releasable pool, while the release probability did not change. Inhibition of calmodulin by W-7 or of calcium/calmodulin-dependent kinase II (CaMKII) by KN-93 completely prevented the potentiating effect of BzATP on the EPP quantal content. The blockade of L-type calcium channels also prevented BzATP action on evoked synaptic activity. Thus, the activation of presynaptic P2X7 receptors in mice lacking pannexin 1 resulted in enhanced evoked ACh release. Such enhanced release was provoked by triggering the calmodulin- and CaMKII-dependent signaling pathway, followed by activation of presynaptic L-type calcium channels. We suggest that in WT mice, this pathway is downregulated due to pannexin 1-dependent tonic activation of inhibitory presynaptic purinergic receptors, which overcomes P2X7-mediated effects.

Ryanodine- and CaMKII-dependent release of endogenous CGRP induces an increase in acetylcholine quantal size in neuromuscular junctions of mice.

OBJECTIVE: The aim of this study was to identify the mechanism responsible for an increase in miniature endplate potentials (MEPPs) amplitude, induced by ryanodine as an agonist of ryanodine receptors in mouse motor nerve terminals. METHODS: Using intracellular microelectrode recordings of MEPPs and evoked endplate potentials (EPPs), the changes in spontaneous and evoked acetylcholine release in motor synapses of mouse diaphragm neuromuscular preparations were studied. RESULTS: Ryanodine (0.1 µM) increased both the amplitudes of MEPPs and EPPs to a similar extent (up to 130% compared to control). The ryanodine effect was prevented by blockage of receptors of calcitonin gene-related peptide (CGRP) by a truncated peptide CGRP8-37 . Endogenous CGRP is stored in large dense-core vesicles in motor nerve terminals and may be released as a co-transmitter. The ryanodine-induced increase in MEPPs amplitude may be fully prevented by inhibition of vesicular acetylcholine transporter by vesamicol or by blocking the activity of protein kinase A with H-89, suggesting that endogenous CGRP is released in response to the activation of ryanodine receptors. Activation of CGRP receptors can, in turn, upregulate the loading of acetylcholine into synaptic vesicles, which will increase the quantal size. This new feature of endogenous CGRP activity looks similar to recently described action of exogenous CGRP in motor synapses of mice. The ryanodine effect was prevented by inhibitors of Ca/Calmodulin-dependent kinase II (CaMKII) KN-62 or KN-93. Inhibition of CaMKII did not prevent the increase in MEPPs amplitude, which was caused by exogenous CGRP. CONCLUSIONS: We propose that the activity of presynaptic CaMKII is necessary for the ryanodine-stimulated release of endogenous CGRP from motor nerve terminals, but CaMKII does not participate in signaling downstream the activation of CGRP-receptors followed by quantal size increase.

Protonophoric action of triclosan causes calcium efflux from mitochondria, plasma membrane depolarization and bursts of miniature end-plate potentials.

The formerly widely used broad-spectrum biocide triclosan (TCS) has now become a subject of special concern due to its accumulation in the environment and emerging diverse toxicity. Despite the common opinion that TCS is an uncoupler of oxidative phosphorylation in mitochondria, there have been so far no studies of protonophoric activity of this biocide on artificial bilayer lipid membranes (BLM). Yet only few works have indicated the relationship between TCS impacts on mitochondria and nerve cell functioning. Here, we for the first time report data on a high protonophoric activity of TCS on planar BLM. TCS proved to be a more effective protonophore on planar BLM, than classical uncouplers. Correlation between a strong depolarizing effect of TCS on bacterial membranes and its bactericidal action on Bacillus subtilis might imply substantial contribution of TCS protonophoric activity to its antimicrobial efficacy. Protonophoric activity of TCS, monitored by proton-dependent mitochondrial swelling, resulted in Ca2+ efflux from mitochondria. A comparison of TCS effects on molluscan neurons with those of conventional mitochondrial uncouplers allowed us to ascribe the TCS-induced neuronal depolarization and suppression of excitability to the consequences of mitochondrial deenergization. Also similar to the action of common uncouplers, TCS caused a pronounced increase in frequency of miniature end-plate potentials at neuromuscular junctions. Thus, the TCS-induced mitochondrial uncoupling could alter neuronal function through distortion of Ca2+ homeostasis.

Calcitonin gene-related peptide increases acetylcholine quantal size in neuromuscular junctions of mice.

We used an intracellular microelectrode technique to study the mechanisms of action of two isoforms (human and rat) of calcitonin gene-related peptide (CGRP) on the evoked and spontaneous quantal secretion of acetylcholine (ACh) in mouse diaphragm motor synapses. Recordings of miniature endplate potentials (MEPPs) and evoked multiquantal endplate potentials (EPPs) in a cut neuromuscular preparation showed that CGRP increased the amplitude of EPPs without influencing their quantal content. Both isoforms of CGRP in a wide range of concentrations (1nM-1µM) provoked a similar considerable increase in MEPPs amplitude in a dose-dependent manner (up to 150-160% compared to control) without changing their frequency, rise-time, and decay. Inhibition of CGRP-receptors by truncated CGRP (CGRP8-37) completely prevented the potentiating effect of CGRP on the MEPPs amplitude. The effect of CGRP was not accompanied by changes in input resistance of muscle fiber membrane but was fully prevented by inhibition of vesicular ACh transport by vesamicol. Inhibition of protein kinase A (PKA) by H-89 also prevented CGRP action on the MEPPs amplitude. It is concluded that, in mammalian neuromuscular junctions, different isoforms of exogenously applied CGRP uniformly potentiate amplitudes of evoked and spontaneous postsynaptic potentials acting presynaptically via an increase in ACh quantal size.