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.

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 wildtype (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 CaMKIIdependent 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.

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The changes in spontaneous and evoked neurotransmitter release caused by agonists and antago nists of the A1 and 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 intra cellular 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 pre vails 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 cal cineurin 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 con clusion, 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 volt age dependent calcium channels → ryanodine receptors of calcium stores. Final effect on the neurotrans mitter release depends on conditions of coactivation of these receptors and interplay of enzymes and L type voltage dependent calcium channels within synaptic terminals.

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.