Reactive oxygen species (ROS) participate in various physiological and pathological processes in the nervous system, but the specific pathways that mediate ROS signalling remain largely unknown. Using electrophysiological techniques and biochemical evaluation of isolated fusion proteins, we explored the sensitivity to standard oxidative stress of the entire synapse, the pre-synaptic machinery and essential fusion proteins underlying transmitter exocytosis. Oxidative stress induced by H 2 O 2 plus Fe 2+ inhibited both evoked and spontaneous quantal release from frog or mouse motor nerve endings, while it left post-synaptic sensitivity unchanged. The depressant effect of H 2 O 2 on acetylcholine release was pertussis toxin-insensitive, ruling out G-protein cascades. Experiments with ionomycin, a Ca 2+ ionophore, revealed that ROS directly impaired the function of releasing machinery. In line with this, SNAP25, one of three essential fusion proteins, showed a selectively high sensitivity to the oxidative signals. Several ROS scavengers enhanced evoked synaptic transmission, consistent with tonic inhibition by endogenous ROS. Our data suggest that ROS-induced impairment of releasing machinery is mediated by SNAP25, which appears to be a pre-synaptic ROS sensor. This mechanism of ROS signalling could have widespread implications in the nervous system and might contribute to the pathogenesis of neurodegenerative diseases.

There is evidence that reactive oxygen species (ROS) are produced and released during neuromuscular activity, but their role in synaptic transmission is not known. Using a two‐electrode voltage‐clamp technique, at frog neuromuscular junctions, the action H2O2 on end‐plate currents (EPC) was studied to determine the targets for this membrane‐permeable ROS. In curarized or cut muscles, micromolar concentrations of H2O2 increased the amplitude of EPCs. Higher (> 30 μm) doses inhibited EPCs and prolonged current decay. These effects were presynaptic since H2O2 did not change the amplitude or duration of miniature EPCs (although it reduced the rate of spontaneous release at high concentrations). Quantal analysis and deconvolution methods showed that facilitation of EPCs was due to increased quantal release, while depression was accompanied by temporal dispersion of evoked release. Extracellular recordings revealed prolonged presynaptic Ca2+ entry in the presence of high H2O2. Both low and high H2O2 increased presynaptic potentiation during high‐frequency stimulation. Pro‐oxidant Fe2+ did not affect facilitation by low doses of H2O2 but augmented the inhibition of EPCs by high H2O2, indicating involvement of hydroxyl radicals. High Mg2+ and the ROS scavenger N‐acetylcysteine eliminated both the facilitatory and depressant effects of H2O2. The facilitatory effect of H2O2 was prevented by protein kinase C (PKC) inhibitors and 4β‐phorbol 12‐myristate, 13‐acetate (PMA), an activator of PKC. PKC inhibitors but not PMA also abolished the depressant effect of H2O2. Our data suggest complex presynaptic actions of H2O2, which could serve as a fast feedback modulator of intense neuromuscular transmission.

During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H 2 O 2 . Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe 2+ . Exogenous H 2 O 2 reproduced the depressant effects of ATP and showed similar sensitivity to anti-and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2 ,7 -dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings.

We have shown previously that ATP inhibits transmitter release at the neuromuscular junction through the action on metabotropic P2Y receptors coupled to specific second messenger cascades. In the present study we recorded K(+) or Ca(2+) currents in motor nerve endings or blocked K(+) or Ca(2+) channels in order to explore the nature of downstream presynaptic effectors. Endplate currents were presynaptically depressed by ATP. Blockers of Ca(2+)-activated K(+)-channels, such as iberiotoxin, apamin or tetraethylammonium, did not change the depressant action of ATP. By contrast, K(+) channel blocker 4-aminopyridine (4-AP) and raised extracellular Ca(2+) attenuated the effect of ATP. However, these effects of 4-AP and high Ca(2+) were reversed by Mg(2+), suggesting Ca(2+)-dependence of the ATP action. Ba(2+) promoted the depressant action of ATP as did glibenclamide, a blocker of ATP-sensitive K(+) channels, or mild depolarization produced by 7.5 mm K(+). None of the K(+) channel blockers affected the depressant action of adenosine. Focal recording revealed that neither ATP nor adenosine affected the fast K(+) currents of the motor nerve endings. However, unlike adenosine, ATP or UTP, an agonist of P2Y receptors, reversibly reduced the presynaptic Ca(2+)-current. This effect was abolished by suramin, an antagonist of P2 receptors. Depressant effect of ATP on the endplate and Ca(2+)-currents was mimicked by arachidonate, which precluded the action of ATP. ATP reduced acetylcholine release triggered by ionomycin or sucrose, suggesting inhibition of release machinery. Thus, the presynaptic depressant action of ATP is mediated by inhibition of Ca(2+) channels and by mechanism acting downstream of Ca(2+) entry.

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