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.