Plant protection products are essential to increase crops quality. However, European and national directives advocate a reduction of their use from 50% with the aim to protect the environment and to limit effect on non-targeted organisms. In this perspective, in order to optimize the efficacy of these products while reducing their dose of use, it is necessary to characterize cellular and intracellular factors which are involved in the modulation of insecticide effect on their membrane target. Among the insecticides most use in crop protection, neonicotinoids rank as one of the most important class in the market. They act on insect nicotinic acetylcholine receptors (nAChRs) from insect central nervous system. From the cockroach Periplaneta americana, neurosecretory cells, identified as Dorsal Unpaired Median (DUM) neurons, express two subtypes of nAChRs (nAChR1 and nAChR2). Whereas nAChR1 is sensitive to imidacloprid (IMI), a first generation of neonicotinoid, this insecticide has no effect on nAChR2. In order to acquire a better understanding of the mode of action of these insecticides on insensitive nAChRs, the aim of this work was to characterize, from an electro-pharmacological point of view, the cellular and molecular factors which influence the efficacy of a second generation neonicotinoid, acetamiprid (ACT) which present a different chemical structure from IMI. Using the patch-clamp technique, under voltage-clamp mode, it was demonstrated that the effect of ACT on nAChR2 is dependent on transmembrane potential. A membrane depolarization slightly increases nAChR2 sensitivity to ACT whereas a hypepolarisation induces an opposite effect. In these two cases, an involvement of intracellular calcium was demonstrated. For potential more positive than the membrane potential (i.e. -50 mV), the inhibition of calcium influx via high-voltage activated (HVA) channels by cadmium chloride and ω-conotoxine GVIA increases the nAChR2 sensitivity to ACT. Similar effects are obtained for membrane potential more hyperpolarized when calcium permeability is inhibited by LOE 908, a specific inhibitor of TRPγ channels. In this case, the use of specific pharmacological tools (forskoline, W7) allowed to reveal the implication of the signaling pathway of cyclicAMP/adenylyl cyclase in the modulation of ACT efficacy on nAChR2. Finally, measurements of membrane resistance, realized at the same time in current-clamp mode, indicate that the conformational state of nAChR2 has an important role in the modulation of nAChR2 sensitivity to ACT. All of results which allowed to identify new cellular and molecular factors involved in the modulation of nAChR2 sensitivity to the neonicotinoid, ACT, open new interesting perspectives in order to optimize the efficacy of insecticide treatment.
