title: Injection of insect membrane in Xenopus oocyte: An original method for the pharmacological characterization of neonicotinoid insecticides.
abstract: Insect nicotinic acetylcholine receptors (nAChRs) represent a major target of insecticides, belonging to the neonicotinoid family. However, the pharmacological profile of native nAChRs is poorly documented, mainly because of a lack of knowledge of their subunit stoichiometry, their tissue distribution and the weak access to nAChR-expressing cells. In addition, the expression of insect nAChRs in heterologous systems remains hard to achieve. Therefore, the structure-activity characterization of nAChR-targeting insecticides is made difficult. The objective of the present study was to characterize insect nAChRs by an electrophysiological approach in a heterologous system naturally devoid of these receptors to allow a molecular/cellular investigation of the mode of action of neonicotinoids. Methods To overcome impediments linked to the expression of insect nAChR mRNA or cDNA, we chose to inject insect membranes from the pea aphid (Acyrthosiphon pisum) into Xenopus oocytes. This microtransplantation technique was designed to gain access to native nAChRs embedded in their membrane, through direct stimulation with nicotinic agonists. Results We provide evidence that an enriched-nAChR membrane allows us to characterize native receptors. The presence of such receptors was confirmed with fluorescent α-BgTX labeling. Electrophysiological recordings of nicotine-induced inward currents allowed us to challenge the presence of functional nAChR. We compared the effect of nicotine (NIC) with clothianidin (CLO) and we assessed the effect of thiamethoxam (TMX). Discussion This technique has been recently highlighted with mammalian and human material as a powerful functional approach, but has, to our knowledge, never been used with insect membrane. In addition, the use of the insect membrane microtransplantation opens a new and original way for pharmacological screening of neurotoxic insecticides, including neonicotinoids. Moreover, it might also be a powerful tool to investigate the pharmacological properties of insect nAChR.
authors.affiliation: Laboratoire de Biologie Neurovasculaire et Mitochondriale Intégrée CNRS UMR6214, INSERM U1083, Univ. Angers Faculté de Médecine, rue Haute de Reculée, 49045 Angers cedex 01, France.
authors.forename: Lucille
authors.initials: L
authors.lastname: Crespin
authors.affiliation: Laboratoire de Biologie Neurovasculaire et Mitochondriale Intégrée CNRS UMR6214, INSERM U1083, Univ. Angers Faculté de Médecine, rue Haute de Reculée, 49045 Angers cedex 01, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Legros
authors.affiliation: Récepteurs et Canaux Ioniques Membranaires, UPRES-EA 2647 USC INRA 1330, SFR 4207 QUASAV, Univ. Angers, 2 Bd Lavoisier, 49045 Angers cedex 01, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: List
authors.affiliation: INRA/Université d'Angers, Neuroéthologie-RCIM, UPRES-EA 2647 USC INRA 1330, SFR 4207 QUASAV, 42, rue Georges Morel, 49071 Beaucouzé, France. Electronic address: helene.leignel@univ-angers.fr.
authors.forename: Hélène
authors.initials: H
authors.lastname: Tricoire-Leignel
authors.affiliation: Laboratoire de Biologie Neurovasculaire et Mitochondriale Intégrée CNRS UMR6214, INSERM U1083, Univ. Angers Faculté de Médecine, rue Haute de Reculée, 49045 Angers cedex 01, France. Electronic address: cesar.mattei@univ-angers.fr.
authors.forename: César
authors.initials: C
authors.lastname: Mattei
mesh_terms: D000818:Animals; D006146:Guanidines / Q000494:pharmacology; D007313:Insecta / Q000187:drug effects* / Q000378:metabolism; D007306:Insecticides / Q000494:pharmacology*; D008566:Membranes / Q000187:drug effects* / Q000378:metabolism; D000073943:Neonicotinoids; D009538:Nicotine / Q000378:metabolism*; D009574:Nitro Compounds / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010078:Oxazines / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013329:Structure-Activity Relationship; D000077922:Thiamethoxam; D013844:Thiazoles / Q000494:pharmacology; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D006146:Guanidines; D007306:Insecticides; D000073943:Neonicotinoids; D009574:Nitro Compounds; D010078:Oxazines; D011978:Receptors, Nicotinic; D013844:Thiazoles; C480342:clothianidin; D009538:Nicotine; D000077922:Thiamethoxam
keywords: Insect membrane; Microtransplantation method; Neonicotinoid insecticides; Nicotinic acetylcholine receptors; Voltage-clamp; Xenopus oocyte