Calcium pathways such as cAMP modulate clothianidin action through activation of a-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors Delphine Calas-List, Olivier List, Sophie Quinchard, Steeve H. Thany * Universite ´ d’Angers, Laboratoire Re ´cepteurs et Canaux Ioniques Membranaires (RCIM), UPRES EA 2647/USC INRA 1330, UFR de Sciences, 2 Bd. Lavoisier, 49045 Angers, France 1. Introduction Insect nicotinic acetylcholine receptors (nAChRs) are penta- meric receptors, formed by the combination of identical (homo- meric) or different (heteromeric) subunits conferring distinct electrophysiological and pharmacological properties (Lansdell et al., 2012; Matsuda et al., 2001; Thany et al., 2007). They are involved in many processes such as rapid synaptic transmission, learning and memory (Gauthier, 2010; Gauthier et al., 2006) and are the main targets of neonicotinoid insecticides (Matsuda et al., 2005; Millar and Denholm, 2007; Thany et al., 2007; Tomizawa and Casida, 2003, 2009). Evidence of neonicotinoids action on insect nAChRs is based in part on electrophysiological studies (Ihara et al., 2007; Liu et al., 2009; Tan et al., 2007). Neonicotinoids such as clothianidin (CLO) are synthetic compounds that have been used as insecticides against agricultural and household pests (Uneme, 2011). They are reported to act as full or partial agonists on insect nAChRs affecting the central nervous system (Deglise et al., 2002; Tan et al., 2007). In our previous studies using cockroach Periplaneta americana, we have demonstrated that application of CLO onto the sixth abdominal ganglion resulted in a dose-dependent depolarization of cockroach cercal afferent/giant interneuron synapses which was not reversible, suggesting a strong desensitization of postsynaptic interneurons (Thany, 2009). CLO-induced currents were insensi- tive to muscarinic antagonist but were blocked by nicotinic antagonists such as a-bungarotoxin (a-Bgt), mecamylamine (Mec) and d-tubocurarine (d-TC). At extrasynaptic level, on dorsal unpaired median (DUM) neurons, CLO current amplitudes were reduced by a-Bgt and completely blocked by MLA, Mec and d-TC (Thany, 2009). Thus, we have suggested that CLO acted as a specific agonist of cockroach a-Bgt-sensitive and -insensitive nAChRs including nAChR1 and nAChR2. We also confirmed that DUM neurons expressed a-Bgt-insensitive nAChR subtypes which were blocked by Mec and d-TC (Calas-List et al., 2012). Two distinct subtypes of a-Bgt-insensitive nAChRs, named nAChR1 and nAChR2, were previously identified (Courjaret and Lapied, 2001). The inward currents mediated by these nAChRs differed from each other on the basis on their voltage dependence (nAChR1 being activated between À80 mV and À30 mV whereas nAChR2 was activated between À30 mV and +20 mV), selective pharma- cological properties (nAChR1 was blocked by d-TC, and nAChR2 NeuroToxicology 37 (2013) 127–133 A R T I C L E I N F O Article history: Received 8 February 2013 Accepted 22 April 2013 Available online 28 April 2013 Keywords: Insect Cockroach DUM neurons Nicotinic receptors Neonicotinoid Clothianidin Calcium cAMP A B S T R A C T Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of a-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors expressed in the cockroach dorsal unpaired median neurons. Clothianidin was able to act as an agonist of imidacloprid-insensitive nAChR2 receptor and internal regulation of cAMP concentration modulated nAChR2 sensitivity to clothianidin. In the present study, we demonstrated that cAMP modulated the agonist action of clothianidin via a-bungarotoxin-sensitive and insensitive receptors. Clothianidin- induced current–voltage curves were dependent to clothianidin concentrations. At 10 mM clothianidin, increasing cAMP concentration induced a linear current–voltage curve. Clothianidin effects were blocked by 0.5 mM a-bungarotoxin suggesting that cAMP modulation occurred through a- bungarotoxin-sensitive receptors. At 1 mM clothianidin, cAMP effects were associated to a- bungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5 mM mecamylamine and 20 mM d-tubocurarine. In addition, we found that application of 1 mM clothianidin induced a strong increase of intracellular calcium concentration. These data reinforced the finding that calcium pathways including cAMP modulated clothianidin action on insect nicotinic acetylcholine receptors. We proposed that intracellular calcium pathways such as cAMP could be a target to modulate the mode of action of neonicotinoid insecticides. ß 2013 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +33 241735213; fax: +33 241735215. E-mail address: steeve.thany@univ-angers.fr (S.H. Thany). Contents lists available at SciVerse ScienceDirect NeuroToxicology 0161-813X/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2013.04.011