Neuroscience Letters 416 (2007) 6–11 A lysolecithin/fatty acid mixture promotes and then blocks neurotransmitter release at the Drosophila melanogaster larval neuromuscular junction Aram Megighian a,∗ , Michela Rigoni b , Paola Caccin b , Mauro A. Zordan c , Cesare Montecucco b a Department of Human Anatomy and Physiology, Section of Physiology, University of Padova, Via Marzolo 3, 35131-Padova, Italy b Department of Biomedical Sciences and Consiglio Nazionale Ricerche Institute of Neuroscience, University of Padova, Italy c Department of Biology, University of Padova, Italy Received 19 September 2006; received in revised form 15 January 2007; accepted 19 January 2007 Abstract The study of the effect of snake presynaptic neurotoxins with phospholipase A2 activity on nerve terminals has recently unveiled the inhibitory action of a lysophosphatidylcholine (LysoPC)/fatty acid mixture. We report here that these neurotoxins have no activity on Drosophila melanogaster nerve terminals. However, a 1:1 mixture of LysoPC and oleic acid induces an early increase, followed by an inhibition of both evoked and spontaneous neurotransmitter release. This effect is also induced by LysoPC alone. The present findings provide an indirect evidence that the lipid hemifusion- to-pore transition is a key event in neuroexocytosis in Drosophila. Moreover, these findings substantiate the use of LysoPC as a general agonist of membrane fusion at nerve terminals. This novel tool could contribute to the unraveling of the molecular steps involved in neuroexocytosis, particularly in Drosophila, where it is straightforward to combine it with electrophysiology and genetics. © 2007 Published by Elsevier Ireland Ltd. Keywords: Neuromuscular junction; Electrophysiology; Snake presynaptic neurotoxins; Lysophosphatidyl choline; Membrane fusion; Exocytosis; Endocytosis Snake presynaptic phospholipase A2 neurotoxins (SPANs) are specific inhibitors of the vertebrate neuromuscular junction and their mechanism of action has been the subject of a large number of studies [13]. The action of these neurotoxins at the neuromus- cular junction is characterized by the induction of acetylcholine release followed by run-down and paralysis of the junction. This is accompanied by enlargement of nerve terminals and depletion of their content of synaptic vesicles (SV) [7,18]. SPANs have been recently studied in primary cultures of different neurons and this approach has revealed that they stimulate exocytosis of synaptic vesicles and at the same time inhibit endocytosis [3,21]. Both these effects are mediated by binding of the neurotoxins to the presynaptic membrane, and hydrolysis of the major phos- pholipid of the membrane outer layer, i.e. phosphatidylcholine, with the production of lysophosphatidylcholine and fatty acids (FA). These lipids influence the local curvature of the mem- brane with promotion of exocytosis and inhibition of endocytosis [3,20]. Here, we have extended these studies to the Drosophila melanogaster larval neuromuscular junction (NMJ) which offers ∗ Corresponding author. Tel.: +39 049 827 5724; fax: +39 049 827 5301. E-mail address: aram.megighian@unipd.it (A. Megighian). the advantage of the availability of several mutants defective in exo-endocytosis and an easier accessibility of the NMJ to exper- imentation [4,8,16,19,24,32]. Surprisingly, we found that none of the four SPANs tested were active in this model system. Thus, we analyzed the effect of LysoPC and FA on evoked and spon- taneous neurotransmitter release at the level of the Drosophila larval NMJ. LysoPC + FA rapidly blocked the synaptic vesicle release of neurotransmitter. This activity was observed both in the presence (evoked release) and in the absence (spontaneous synaptic release) of nerve stimulation. A similar inhibition was caused by LysoPC alone. Canton S flies were raised on a standard yeast–glucose–agar medium [23] and maintained at 23 ◦ C, 70% relative humidity and in 12-h light:12-h dark cycles. Experiments were performed at 20–22 ◦ C on Drosophila third instar larval body-wall dissected in Ca 2+ free HL3 saline [25] and pinned on the sylgard (Sylgard 184, Dow Corning) coated surface of a 35 mm Petri dish [2]. For electrophysiologi- cal experiments, Ca 2+ free HL3 was replaced with 1 ml Ca 2+ 0.4 mM HL3 in order to minimize muscle contractions dur- ing segmental nerve stimulation. Single segmental nerves were stimulated (square wave stimuli, 0.15 ms duration × 1.5 thresh- old voltage) using a suction electrode connected to a Grass 0304-3940/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2007.01.040