Mouse Motor Synapses Research Articles

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 (30Hz, 2min) 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.

Noncanonical Potentiation of Evoked Quantal Release of Acetylcholine by Cannabinoids Anandamide and 2-Arachidonoylglycerol in Mouse Motor Synapses

Noncanonical Potentiation of Evoked Quantal Release of Acetylcholine by Cannabinoids Anandamide and 2-Arachidonoylglycerol in Mouse Motor Synapses

Acetylcholine Release in Mouse Motor Synapses. Changes of Purinergic Regulation under Conditions of Pharmacological Blockade of Pannexin 1 and Its Genetic Knockout

Acetylcholine Release in Mouse Motor Synapses. Changes of Purinergic Regulation under Conditions of Pharmacological Blockade of Pannexin 1 and Its Genetic Knockout

Spontaneous Acetylcholine Release Potentiation Induced by 2-Arachidonoylglycerol and Anandamide in Mouse Motor Synapses

Spontaneous Acetylcholine Release Potentiation Induced by 2-Arachidonoylglycerol and Anandamide in Mouse Motor Synapses

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.

Mechanism of Anandamide Action on Spontaneous Acetylcholine Release in Mouse Motor Synapses

Mechanism of Anandamide Action on Spontaneous Acetylcholine Release in Mouse Motor Synapses

The Participation of Endocannabinoid Receptors in the Regulation of Spontaneous Synaptic Activity at Neuromuscular Junctions of Mice

The mechanisms underlying changes in spontaneous release of acetylcholine (ACh) induced by exogenous agonists and antagonists of endocannabinoid receptors were investigated by recording miniature endplate potentials (MEPPs) in motor synapses of mouse diaphragm preparations with intracellular microelectrodes. There have not been observed any statistically significant changes either in the membrane potential of muscle fibers or in the frequency, amplitude, and time parameters of the MEPPs under the action of AM-251 (1 μM), antagonist/inverse agonist of CB1 receptors, for 60 min. Activation of CB receptors by their exogenous agonist WIN 55,212-2 (WIN) (20 μM) for 1 h did not cause statistically significant changes in the amplitude of the MEPPs. However, MEPP frequency increased by more than 50% as compared to the control under the action of WIN for 30–60 min. Blocking of CB1 receptors by AM-251 (1 μM) prevented the WIN-induced increase in MEPP frequency. A protein kinase A inhibitor H-89 (1 μM) did not prevent the enhancement of the ACh secretion upon activation of CB receptors by WIN. WIN lost the ability to increase MEPP frequency in the presence of a phospholipase C inhibitor U73122 (5 μM), which did not affect by itself the amplitude and temporal characteristics or MEPP frequency. A protein kinase C inhibitor, chelerythrin (4 μM) or ryanodine, which blocks the ryanodine receptors (RyRs) at a concentration of 5 μM, also prevented the WIN-induced increase in MEPP frequency. Thus, a signaling cascade triggered by stimulation of presynaptic CB1 receptors with subsequent activation of phospholipase C, protein kinase C, and the release of stored calcium, leading to an increase in the frequency of spontaneous quantal release of ACh, has been detected for the first time in mouse motor synapses. This suggests the presence of CB1 receptors on the presynaptic membrane in mouse motor nerve terminals and the possibility of activation of these receptors by endocannabinoids for potentiating the spontaneous secretion of ACh.

The Role of BDNF and proBDNF in the Regulation of Parameters of Spontaneous Acetylcholine Secretion in Mature and Newly-Formed Mouse Motor Synapses

The Role of BDNF and proBDNF in the Regulation of Parameters of Spontaneous Acetylcholine Secretion in Mature and Newly-Formed Mouse Motor Synapses

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.

The Participation of Presynaptic Alpha7 Nicotinic Acetylcholine Receptors in the Inhibition of Acetylcholine Release during Long-Term Activity of Mouse Motor Synapses

In the motor synapses of the mouse diaphragm, we recorded the miniature endplate potentials (MEPPs) and multiquantal endplate potentials induced by stimulation of the phrenic nerve (EPPs). Prolonged continuous rhythmic stimulation (50 Hz for 40 seconds) caused a depression of the synaptic transmission in the form of a gradual biphasic decrease in the quantal content of EPPs in the train. A rapid decrease in the quantal content of the EPPs to 50% of the amplitude of the first EPPs in the train (EPP1) during the first 10 seconds of activity was followed by a slower decrease in the EPP quantum content to 35–40% of EPP1 by the 40th second of stimulation. Blockage of the α7 nicotinic acetylcholine receptors by methyllycaconitine (20 nM), as well as ryanodine receptors by ryanodine (3 µM), or small-conductance calcium-activated potassium channels of the SK-type by apamin (1 µM) significantly reduced the development of depression; the EPP quantal content decreased to 65–70% of EPP1 by the 10th second of stimulation and maintained at this level for the next 30 seconds of stimulation. It was concluded that in mouse cholinergic motor synapses, the mechanism of transient depression of transmission may be autoinhibition of acetylcholine quantal secretion by endogenous acetylcholine/choline, which activates the α7 nicotinic acetylcholine receptors and triggers the signaling cascade that involves presynaptic ryanodine receptors and SK channels.

CaMKII Is Involved in the Choline-Induced Downregulation of Acetylcholine Release in Mouse Motor Synapses

We investigated the involvement of calcium-dependent enzymes, protein kinase C (PKC) and calcium-calmodulin-dependent protein kinase II (CaMKII), in the signaling pathway triggered by the activation of presynaptic alpha7-type nicotinic acetylcholine receptors by exogenous choline, leading to downregulation of the evoked acetylcholine (ACh) release in mouse motor synapses. Blockade of PKC with chelerythrine neither changed the evoked release of ACh by itself nor prevented the inhibitory effect of choline. The CaMKII blocker KN-62 did not affect synaptic activity but fully prevented the choline-induced downregulation of ACh release.

CaMKII участвует в вызванном холином торможении секреции ацетилхолина в моторных синапсах мыши

CaMKII участвует в вызванном холином торможении секреции ацетилхолина в моторных синапсах мыши

CaMKII участвует в вызванном холином торможении секреции ацетилхолина в моторных синапсах мыши

We investigated the involvement of calcium-dependent enzymes, protein kinase C (PKC) and calcium-calmodulin-dependent protein kinase II (CaMKII), in the signaling pathway triggered by the activation of presynaptic alpha7-type nicotinic acetylcholine receptors by exogenous choline, leading to downregulation of the evoked acetylcholine (ACh) release in mouse motor synapses. Blockade of PKC with chelerythrine neither changed the evoked release of ACh by itself nor prevented the inhibitory effect of choline. The CaMKII blocker KN-62 did not affect synaptic activity but fully prevented the choline-induced downregulation of ACh release.

The role of pannexin 1 in the purinergic regulation of synaptic transmission in mouse motor synapses

The role of pannexin 1 in the release to the extracellular space of ATP/adenosine modulating the acetylcholine (ACh) secretion was studied in mouse diaphragm motor synapses. Using neuromuscular preparations obtained from wild-type and pannexin-1 knockout mice, the miniature endplate potential (MEPPs) and evoked endplate potentials (EPPs) were recorded in combination with pharmacological modulation of P2-type ATP receptors and A1-type adenosine receptors. Selective inhibition of A1 receptors with DPCPX or P2 receptors with PPADS increased quantal content of EPPs in wild-type mice. MRS 2211, selective antagonist of P2Y13 receptors, produced the same effect. Activation of receptors A1 or P2Y13 by their agonists (2-CADO and IDP, respectively) decreased the EPP quantal content. It means that the activity of endogenous ATP and adenosine is synergistic and directed to depression of the ACh release. ARL67156, an inhibitor of synaptic ecto-ATPases, which blocks the hydrolysis of ATP to adenosine and increases the level of ATP in the synaptic cleft, prolonged EPPs without changing their quantal content. In pannexin-1 knockout mice there were no changes in the EPP quantal content and in other parameters of synaptic transmission as compared to wildtype mice. However, downregulation of purinergic effects with antagonists of A1 or P2 receptors (DPCPX, PPADS, MRS 2211) did not change EPP quantal content and any other parameters of spontaneous or evoked ACh release in all cases. ARL67156 did not alter the temporal parameters of EPPs, either. Nevertheless, 2-CADO, the A1-type receptor agonist, decreased the EPP quantal content, while the agonist of P2Y13 receptors decreased the MEPP amplitude. Thus, in mice lacking pannexin 1, procedures revealing the presence and regulatory activity of synaptic ATP/adenosine did not change the parameters of synaptic transmission. The obtained data substantiate a mandatory role of pannexin 1 in the purinergic regulation of motor synapse activity by endogenous ATP/adenosine.

The role of adenosine receptors and L-type calcium channels in the regulation of the mediator secretion in mouse motor synapses

The changes in spontaneous and evoked neurotransmitter release caused by agonists and antagonists of the A1and A2A-subtypes of adenosine receptors combined with inhibition of some enzymes and voltage-dependent calcium channels of L-type were studied in mouse diaphragm motor synapses using intracellular microelectrode recordings of miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs). Simultaneous activation of presynaptic A1 and A2A receptors by endogenous adenosine during short-term rhythmic activity of motor synapses was shown for the first time. Activation of receptors A1 prevails and is followed by downregulation of the ACh release due to inhibition of intracellular cascade, which involves protein kinase A (PKA) and L-type voltage-dependent calcium channels. Activation of receptors A2A with their agonist CGS-21680 caused upregulation of the ACh secretion due to enhancement of PKA activity followed by activation of L-type voltage-dependent calcium channels. The mechanism of the evoked release potentiation, when A1 receptors are blocked or the activity of A2A receptors prevails over that of A1 receptors, involves calcium release from ryanodine-sensitive intracellular calcium stores coupled with PKA and activation of L-type voltage-dependent calcium channels. It was found that protein phosphatase calcineurin participates in downregulation of the L-type voltage-dependent calcium channels irrespective of the A1 receptors. It was shown for the first time that disinhibition of L-type voltage-dependent calcium channels caused by the calcineurin inhibition requires participation of the activity of A2A receptors and PKA. In conclusion, reciprocal interactions between presynaptic receptors A1 and A2A and their effect on the ACh release were shown in motor synapses. These interactions are mediated by the following cascade: PKA → L-type voltage-dependent calcium channels → ryanodine receptors of calcium stores. Final effect on the neurotransmitter release depends on conditions of coactivation of these receptors and interplay of enzymes and L-type voltage-dependent calcium channels within synaptic terminals.

Postsynaptic Potentiation in Mouse Motor Synapses Induced by ATP Accumulation in Synaptic Cleft.

In mouse motor synapses, a non-selective purinoceptor antagonist suramin increased the quantum content of endplate potentials (EPP) without changing the time course of synaptic potentials. An ectonucleotidase inhibitor ARL 67156 had no effect on the amplitude and quantum content of EPP and miniature endplate potentials (mEPP) evoked by single stimuli, but significantly prolonged their duration. Long-term high-frequency stimulation of the nerve in the presence of ARL 67156 persistently increased the amplitude and duration of EPP during the train of impulses, but did not change their quantum content. ATP-γ-S, a non-hydrolyzed ATP analogue, significantly increased the amplitudes and prolonged the rising and falling phases of EPP and mEPP. The ATP-induced postsynaptic potentiation in neuromuscular transmission can result from the increase in ATP content and its longer presence in the synaptic cleft.

The Mechanism of Choline-Mediated Inhibition of Acetylcholine Release in Mouse Motor Synapses

The mechanism of action of tonically applied choline, the agonist of α7 nicotinic acetylcholine receptors (nAChRs), to the spontaneous and evoked release of a neurotransmitter in mouse motor synapses in diaphragm neuromuscular preparations using intracellular microelectrode recordings of miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) was studied. Exogenous choline was shown to exhibit a presynaptic inhibitory effect on the amplitude and quantal content of EPPs for the activity of neuromuscular junction evoked by single and rhythmic stimuli. This effect was inhibited either by antagonists of α7-nAChRs, such as methyllycaconitine and α-cobratoxin, or by blocking SK-type calcium-activated potassium (KCa) channels with apamin or blocking intraterminal ryanodine receptors with ryanodine. A hypothesis was put forward that choline in mouse motoneuron nerve terminals can activate presynaptic α7-nAChRs, followed by the release of the stored calcium through ryanodine receptors and activation of SK-type KCa channels, resulting in sustained decay of the quantal content of the evoked neurotransmitter release.

Involvement of Basal and Calcium-Activated Protein Kinase C in Neurotransmitter Secretion in Mouse Motor Synapses

Blocker of presynaptic protein kinase C isoforms, GF109203X, reduced quantal content of single and rhythmic evoked end-plate potentials. The increase in quantal content of single potentials under the effect of 4- aminopyridine was neutralized by 75% under the effect of L-type Ca(2+)-channel blocker nitrendipine and completely returned to the control level after protein kinase C inhibition with chelerythrine. Neither nitrendipine, nor GF109203X affected the potentiating effect of tetraethylammonium on quantal content of end-plate potentials. Thus, we discovered basal activity of presynaptic protein kinase C under normal conditions that is aimed at the maintenance of quantal content of evoked release. It has been concluded that 4-aminipyridine, but not tetraethylammonium, triggers Ca(2+) entry into the terminal, which activates protein kinase C and enhanced the evoked acetylcholine release.

Facilitation of Neurotransmitter Release in Mouse Motor Synapses in Different Modes of Protein Kinase C Activation

Protein kinase C blocker chelerythrine prevented the increase in quantal content of single and rhythmic evoked end-plate potentials after disinhibition of L-type Ca(2+)-channels with paxillin. Phorbol ester increased quantal content of single end-plate potentials and changed rhythmic activity of mouse motor synapses. The effects of phorbol ester were to a great extent neutralized by L-type Ca(2+)-channel blocker nitrendipine and were completely abolished by K(+)-channel blocker 4-aminopyridine. Thus, we discovered different facilitations of transmission after protein kinase C activation with calcium current through L-type channels and with phorbol ester.

Facilitation of Acetylcholine Secretion in Mouse Motor Synapses Caused by Calcium Release from Depots upon Activation of L-Type Calcium Channels

Pharmacological disinhibition of L-type Ca(2+) channels by two ways (with agonist S(-) BAY K 8644 and iberiotoxin, a Ca(2+)-activated BK-type K(+)-channel blocker) increases quantal content of evoked end-plate potentials, which was completely prevented by ryanodine (2 microM) blockade of ryanodine receptors. We conclude that increased quantal secretion of the transmitter induced by L-type Ca(2+) channel functioning requires activation of ryanodine receptors and calcium release from depots in motor terminals in mice.