15Nicotinic acetylcholine receptors (nAChRs) are the main target of neonicotinoid insecticides, 16 which are widely used in crop protection against insect pests. Electrophysiological and 17 molecular approaches have demonstrated the presence of several nAChR subtypes with 18 different affinities for neonicotinoid insecticides. However, the precise mode of action of 19 neonicotinoids on insect nAChRs remains to be elucidated. Radioligand binding studies with 20 [ 3 H]-α-bungarotoxin and [ 3 H]-imidacloprid have proved instructive in understanding ligand 21 binding interactions between insect nAChRs and neonicotinoid insecticides. The precise 22 binding site interactions have been established using membranes from whole body and specific 23 tissues. In this review, we discuss findings concerning the number of nAChR binding sites 24 against neonicotinoid insecticides from radioligand binding studies on native tissues. We 25 summarize the data available in the literature and compare the binding properties of the most 26 commonly used neonicotinoid insecticides in several insect species. Finally, we demonstrate 27 that neonicotinoid-nAChR binding sites are also linked to biological samples used and insect 28 species. 29 30 31 32 33 Nicotinic acetylcholine receptors (nAChRs) are involved in rapid neurotransmission in 34 both insect and mammalian nervous systems and play major roles in learning and memory [1-35 3]. Because of these central roles, they are the main target of neonicotinoid insecticides which 36are used as a chemical method worldwide to control insect pest [4]. However, this has led to 37 the evolution of resistance resulting in a reduction in effectiveness [5][6][7][8], environmental 38 concerns linked to the accumulation of these compounds and potential effects on non-target 39 insects such as pollinators [9][10][11][12]. Currently, binding studies are used to monitor and analyze 40 the mode of action of neonicotinoid insecticides on insect native nAChRs in order to understand 41 the levels of resistance. Binding studies, as well as the use of electrophysiology, have proven 42 instructive in identifying different nAChR subtypes as well as providing insights into their 43 pharmacological properties. For instance, studies using the patch clamp method demonstrated 44 that imidacloprid (IMI), the forerunner of neonicotioid insecticides, is a partial agonist of insect 45 nAChRs [13-16] while clothianidin (CLT) and acetamiprid (ACE) appear to be full agonists 46 [17]. Moreover, as it is the case with vertebrates, it is possible to identify insect α-bungarotoxin 47 (α-Bgt)-sensitive and -insensitive nAChR subtypes through binding studies [16,[18][19][20]. α-Bgt 48 is a snake toxin commonly used in vertebrates to characterize homomeric nAChRs such as α7 49 receptors [21-23] even though several studies have demonstrated that it can bind to heteromeric 50 α9α10 and homomeric α8 receptors [24,25]. In insect species, CLT binds to both α-Bgt-51 sensitive and -insensitive receptors expressed in the cockroach d...

Neonicotinoid insecticides are used worldwide and have been demonstrated as toxic to beneficial insects such as honeybees. Their effectiveness is predominantly attributed to their high affinity for insect neuronal nicotinic acetylcholine receptors (nAChRs). Mammalian neuronal nAChRs are of major importance because cholinergic synaptic transmission plays a key role in rapid neurotransmission, learning and memory processes, and neurodegenerative diseases. Because of the low agonist effects of neonicotinoid insecticides on mammalian neuronal nAChRs, it has been suggested that they are relatively safe for mammals, including humans. However, several lines of evidence have demonstrated that neonicotinoid insecticides can modulate cholinergic functions through neuronal nAChRs. Major studies on the influence of neonicotinoid insecticides on cholinergic functions have been conducted using nicotine low-affinity homomeric α7 and high-affinity heteromeric α4β2 receptors, as they are the most abundant in the nervous system. It has been found that the neonicotinoids thiamethoxam and clothianidin can activate the release of dopamine in rat striatum. In some contexts, such as neurodegenerative diseases, they can disturb the neuronal distribution or induce oxidative stress, leading to neurotoxicity. This review highlights recent studies on the mode of action of neonicotinoid insecticides on mammalian neuronal nAChRs and cholinergic functions.

This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

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Understanding insect nicotinic acetylcholine receptor (nAChR) subtypes is of major interest because they are the main target of several insecticides. In this study, we have cloned a cockroach Pameα7 subunit that encodes a 518 amino acid protein with futures typical of nAChR subunit, and sequence homology to α7 subunit. Pameα7 is differently expressed in the cockroach nervous system, in particular in the antennal lobes, optical lobes and the mushroom bodies where specific expression was found in the non-compact Kenyon cells. In addition, we found that cockroach Pameα7 subunits expressed in Xenopus laevis oocytes can assemble to form homomeric receptors. Electrophysiological recordings using the two-electrode voltage clamp method demonstrated that nicotine induced an I max current of −92 ± 27 nA at 1 mM. Despite that currents are low with the endogenous ligand, ACh, this study provides information on the first expression of cockroach α7 homomeric receptor.

The functional expression of the cockroach Pameα7 nicotinic acetylcholine receptor subunit has been previously studied, and was found to be able to form a homomeric receptor when expressed in Xenopus laevis oocytes. In this study, we found that the neonicotinoid insecticide imidacloprid is unable to activate the cockroach Pameα7 receptor, although thiacloprid induces low inward currents, suggesting that it is a partial agonist. In addition, the co-application or 5 min pretreatment with 10 µM imidacloprid increased nicotine current amplitudes, while the co-application or 5 min pretreatment with 10 µM thiacloprid decreased nicotine-evoked current amplitudes by 54% and 28%, respectively. This suggesting that these two representatives of neonicotinoid insecticides bind differently to the cockroach Pameα7 receptor. Interestingly, the docking models demonstrate that the orientation and interactions of the two insecticides in the cockroach Pameα7 nAChR binding pocket are very similar. Electrophysiological results have provided evidence to suggest that imidacloprid and thiacloprid could act as modulators of the cockroach Pameα7 receptors.

Insect resistance mechanisms against pesticides lead to the development and the search of new pesticide combinations in order to delay the resistance. The combination of neonicotinoids with pyrethroids was currently proposed but the mode of action of these compounds at synaptic and extrasynaptic levels needs to be further explored. In the present study, we evaluated the effect of the combination of two insecticides, permethrin and dinotefuran, on cockroach cholinergic synaptic transmission and on isolated cell bodies. We first found that combination of 5 μM permethrin and dinotefuran enhances depolarization of the sixth abdominal ganglion compared to dinotefuran alone, without an inhibition of the spontaneous activity. However, a pretreatment with 1 μM dinotefuran or permethrin before bath application of the mixture inhibits the ganglionic depolarization. Compared to permethrin, 1 μM dinotefuran induces a persistent enhancement of spontaneous activity. Interestingly, at extrasynaptic level, using dorsal unpaired median neurons and Kenyon cells, we found that combination of both 1 μM dinotefuran and permethrin resulted in an increase of the mixture-induced current amplitudes. Pretreatment with 1 μM dinotefuran strongly decreases the currents whereas permethrin induces a time-dependent inhibition. These data demonstrate that the combination of dinotefuran and permethrin enhances the effect of dinotefuran.

Nicotinic acetylcholine receptors are an important class of excitatory receptors in the central nervous system of arthropods. In the ticks Ixodes ricinus , the functional and pharmacological properties of nicotinic receptors located in their neurons are still unknown. The objective of this study was to characterize the pharmacological properties of tick nicotinic receptors using membrane microtransplantation in Xenopus laevis oocytes and two-electrodes voltage clamp method. The membranes microtransplanted were extracted from the tick synganglion. We found that oocytes microtransplanted with tick synganglion membranes expressed nicotinic acetylcholine receptor subtypes which were activated by acetylcholine (1 mM) and nicotine (1 mM). Currents induced by pressure application of acetylcholine and nicotine were diminished by 10 nM α-bungarotoxin and methyllycaconitine, suggesting that they expressed two subtypes of nicotinic receptors, α-bungarotoxin-sensitive and -insensitive, respectively. In addition, we found that nicotine receptors expressed in the synganglion membranes were poorly sensitive to the neonicotinoid insecticides clothianidin (CLT), imidacloprid (IMI), acetamiprid (ACE) and thiamethoxam (TMX), in agreement with their lack of activity as acaricides. Interestingly, current amplitudes were strongly potentialized in the presence of 1 μM PNU-120596. CLT was more active as an agonist than IMI, TMX and ACE. Finally, we demonstrated that microtransplantation of purified membrane from the tick synganglion can be a valuable tool for the development and screening of compounds targeting tick nicotinic acetylcholine receptor subtypes.