Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

The role of cGMP-dependent pathways in synaptic vesicle recycling in motor nerve endings during prolonged high-frequency stimulation was studied at frog neuromuscular junctions using electrophysiological and fluorescent methods. An increase of intracellular cGMP concentration (8-Br-cGMP or 8-pCPT-cGMP) significantly reduced the cycle time for synaptic vesicles through the enhancement of vesicular traffic rate from the recycling pool to the readily releasable pool and acceleration of fast endocytosis. Pharmacological inhibition of soluble guanylate cyclase or protein kinase G slowed down the rate of recycling as well as endocytosis of synaptic vesicles. The results suggest that cGMP-PKG-dependent pathway serves a significant function in the control of vesicular cycle in frog motor terminals.

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 H₂O₂ plus Fe²⁺ 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 H₂O₂ on acetylcholine release was pertussis toxin‐insensitive, ruling out G‐protein cascades. Experiments with ionomycin, a Ca²⁺ 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.

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 H₂O₂. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro‐oxidant Fe²⁺. Exogenous H₂O₂ 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.

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 H₂O_2, 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.