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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2017 Oct 6;175(11):1987–1998. doi: 10.1111/bph.14018

Neonicotinoid insecticides differently modulate acetycholine‐induced currents on mammalian α7 nicotinic acetylcholine receptors

Alison Cartereau 1, Carine Martin 1, Steeve H Thany 1,✉
PMCID: PMC5978969  PMID: 28853147

Abstract

Background and Purpose

Neonicotinoid insecticides are described as poor agonists of mammalian nicotinic ACh receptors. In this paper, we show that their effects on mammalian nicotinic receptors differ between compounds.

Experimental Approach

Two‐electrode voltage‐clamp electrophysiology was used to characterize the pharmacology of three neonicotinoid insecticides on nicotinic α7 receptors expressed in Xenopus oocytes. Single and combined application of clothianidin, acetamiprid and thiamethoxam were tested.

Results

Two neonicotinoid insecticides, clothianidin and acetamiprid, were partial agonists of mammalian neuronal α7 nicotinic receptors, whereas another neonicotinoid insecticide, thiamethoxam, which is converted to clothianidin in insect and plant tissues, had no effect. Pretreatment with clothianidin and acetamiprid (10 μM) ACh significantly enhanced the subsequent currents evoked by ACh (100 μM ) whereas pretreatment with thiamethoxam (10 μM) reduced ACh‐induced current amplitudes.A combination of the three neonicotinoids decreased the ACh‐evoked currents.

Conclusions and Implications

The present findings suggest that neonicotinoid insecticides differ markedly in their direct effects on mammalian α7 nicotinic ACh receptors and can also modulate ACh‐induced currents. Furthermore, our data indicate a previously unknown modulation of mammalian α7 nicotinic receptors by a combination of clothianidin, acetamiprid and thiamethoxam.

Linked Articles

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

Abbreviations

nAChR

nicotinic ACh receptor

SOS

standard oocyte saline

Introduction

Neuronal http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=76 (nAChRs) are ligand‐gated ion channels that are located at pre‐ and postsynaptic sites within the central and peripheral nervous system (Wonnacott, 1997; Rousseau et al., 2005). These pentameric receptors are generated from different sets of subunits, and the various subunit combinations differ in their pharmacological and kinetic properties and also in their cellular and subcellular localization in the brain (Gotti et al., 2006; Gotti et al., 2009). The α7 homo‐oligomer is one of the main types of nAChRs in the brain, widely distributed and characterized by a fast activation, a low affinity and a high calcium permeability (Millar and Gotti, 2009; Taly et al., 2009). Several reports indicate that http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=468 are implicated in the pathophysiology of neurodegenerative diseases (Paterson and Nordberg, 2000). The involvement of the α7 nAChRs in neurodegenerative diseases is in part due to their high expression in brain regions that are involved in cognitive processes (Levin et al., 2006). Thus, α7 nAChRs and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4865 are co‐localized in cortical regions of patients with Alzheimer's disease (Wang et al., 2000a,b, 2003; Pym et al., 2005, 2007).

Because of the neuronal importance of α7 nAChRs, they are currently used as tools to identify and characterize the mode of action of diverse compounds that can act as agonists, antagonists and modulators of neuronal nAChRs (Davies et al., 1999; Collins and Millar, 2010; Gill et al., 2013; Echeverria et al., 2016). For example, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2373, a macrocyclic lactone, used commercially as an antiparasitic agent in both human and veterinary medicine, was not able, by itself, to activate currents but acted as a positive allosteric modulator of α7 nAChRs (Krause et al., 1998; Burkhart, 2000; Collins and Millar, 2010). Indeed, ivermectin displayed no agonist activity on rat α7 nAChRs expressed in Xenopus oocytes but caused a potentiation of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=294‐induced responses after pretreatment (Krause et al., 1998; Sattelle et al., 2009). These data added to our understanding of the observed diversity of ivermectin actions on vertebrate and invertebrate ligand‐gated ion channels.

Among the large number and variety of compounds binding to vertebrate and invertebrate nAChRs, neonicotinoid insecticides are of special interest. Indeed, in 2011, the market share of neonicotinoids in the total global market for insecticides was 28.5% (US $12.75 million) (Jeschke et al., 2013). They have no cross‐resistance to conventional, longer established insecticide classes and are replacing the older and environmentally less benign classes, such as pyrethroids, organophosphates, carbamates and several other classes of insecticides used in agriculture. Neonicotinoid insecticides act as agonists of insect neuronal nAChRs with much higher affinity than that of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2585 and ACh. They are considered more toxic to insects (Tomizawa and Casida, 2005; Tomizawa and Casida, 2003) because of their chemical structures, their binding site interactions at the corresponding insect nAChRs and their hydrophobicity, which improves the penetrability of insect integument, enhancing insecticidal efficacy (Tomizawa and Casida, 2009; Tomizawa et al., 2011). Indeed, binding studies reveal that imidacloprid and the parent compounds bound with very low affinities to the rat brain membranes and neuronal α7 and α4β2 nAChRs (Kagabu et al., 2000; Lansdell and Millar, 2000). Moreover, currents elicited by ACh are differently potentiated by clothianidin and imidacloprid suggesting that the effects of neonicotinoids on ACh‐induced currents could be dependent on the chemical structure of neonicotinoids (Toshima et al., 2008; Li et al., 2011).

In the present study, we investigated the mode of action of three different neonicotinoids, clothianidin, acetamiprid and thiamethoxam on rat α7 neuronal nAChRs. These three compounds are members of a group of seven major commercial neonicotinoid insecticides used worldwide, the others being imidacloprid, thiacloprid, dinotefuran and nitenpyram (Jeschke et al., 2013). In addition, we wanted to investigate whether clothianidin, acetamiprid and thiamethoxam in low concentrations, impaired ACh‐induced currents in mammalian α7 receptors. We demonstrated that clothianidin and acetamiprid were partial agonists of α7 nAChRs whereas thiamethoxam had no agonist effect. Also, the three compounds were able to modulate ACh‐evoked currents in different ways.

Methods

Preparation of cRNA

Rat neuronal α7 nAChR (GenBank accession number: S53987) and human RIC3 (GenBank accession number: AY326435) clones were obtained from Prof. Roger Papke (University of Florida) and Prof. Millet Treinin (University of Jerusalem). Each cDNA was cloned into the pGem vector. Recombinant plasmid containing rat α7 nAChR and RIC3 were linearized with Smal (New England Biolabs, Ipswich, USA) or NheI (Promega, Madison, USA) respectively. Capped RNAs were transcribed in vitro using T7 mMESSAGE mMACHINE kit (Ambion, Austin, USA). After linearization and purification of cloned cDNAs, RNA transcripts of α7 and RIC3 were injected in the oocytes for electrophysiological recordings. Xenopus laevis oocytes were obtained from the CRB Xenope, University of Rennes, France. The CRB Xenope is a French national platform dedicated to Xenopus breeding for experimental research. Experiments were conducted during 2 years, and at least 10 female frogs (between 10 and 12 frogs) were used for the studies. Thus, the number of batches (frogs) ‘n’ for each experimental group or condition is indicated and, except where indicated, a minimum of 120 oocytes per batch were recorded for each experimental condition.

Oocyte injection

Oocytes were kept in standard oocyte saline (SOS) of the following composition: in mM, 100 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2 and 5 HEPES, pH 7.5. Stages V and VI oocytes were defolliculated manually using fine forceps, after treatment with 2 mg·mL−1 collagenase IA (Sigma, France) in Ca2+‐free SOS solution, supplemented with 0.8 mg·mL−1 trypsin inhibitor. Defolliculated oocytes were injected in the vegetal pole with approximately 50 nL (3–6 ng per oocytes) (Papke et al., 2004) capped RNA and maintained at 19°C in sterile SOS solution supplemented with penicillin (100 U·mL−1), streptomycin (100 mg·mL−1), gentamycin (50 mg·mL−1) and sodium pyruvate (2.5 mM). This medium was replaced at 24 h intervals.

Voltage‐clamp recordings

Two‐electrode voltage clamp electrophysiology was conducted using DAGAN TEV‐200A amplifier (Dagan Corporation, Minneapolis, USA). Membrane currents were recorded between 4 and 7 days after injection (Papke et al., 2004), using two microelectrodes filled with 3 M KCl and had a resistance ranging between 0.2 and 5 MΩ. The oocyte membrane potential was held at −60 mV and perfused continuously with recording buffer at room temperature (20–22°C). To obtain dose–response relations, the neurotransmitters were applied during 5 s, at 5–10 min intervals. To evaluate synergic effects, the neonicotinoids were applied during 4–5 min before the co‐application with ACh. Experimental data were digitized with a Digidata‐1322A A/D converter and later analysed with pCLAMP (Molecular Devices, Union City, CA, USA). All agonist preparations were prepared with recording buffer. The EC50 values were determined using nonlinear regression on normalized data (1 mM ACh as maximal response) using GraphPad Prism software.

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). All data were collected and analysed without knowledge of the treatments (blinded). Data were shown as mean ± SD and analysed using Prism 7 (GraphPad Software, La Jolla, CA, USA). Responses to experimental neonicotinoid applications were determined relative to the preceding ACh control responses in the same experimental conditions, in order to normalize the data, compensating for the varying levels of channel expression among the oocytes (Papke et al., 2004). The dose–response curves were derived from the fitted curve following the equation: Y = Imin + (Imax – Imin)/(1 + 10(log(EC50−X)H) where Y is the normalized response, Imax and Imin are the maximum and minimum responses, H is the Hill coefficient, EC50 is the concentration giving half the maximum response and X is the logarithm of the compound concentration. In the experiments in which neonicotinoids were used in combination, we used ‘effect‐based strategy’ method, in particular, the Bliss independence model (see Zhao et al., 2014; Foucquier and Guedj, 2015). For each condition, the combination index (CI), which was the standard measure of combination effect, was determined. CI = (EA + EB – EAEB)/EAB, where EA and EB represent two drugs A and B, with respective effects EA and EB, and of combined effect EAB. Thus, CI values >1 indicates antagonism, CI values <1 synergism and CI values = 1, an additive effect (Zhao et al., 2014; Foucquier and Guedj, 2015). Differences between group means were tested for significance by the Kruskal–Wallis one‐way ANOVA on ranks followed by a modified Student's t‐test (Bonferroni post hoc test) in the case of single and multiple comparisons respectively. P < 0.05 was the accepted minimum level of significance.

Materials

ACh, nicotine, clothianidin, acetamiprid and thiamethoxam were purchased from Sigma (St Quentin, France).

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org/, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Results

Clothianidin and acetamiprid act as partial agonists of α7 nicotinic ACh receptors and increase ACh‐induced currents

For better understanding of the mode of action of the three neonicotinoid insecticides (Figure 1A), they were first applied to Xenopus oocytes expressing rat neuronal α7 homomeric nAChRs. Bath application of 1 mM clothianidin and acetamiprid induced inward currents, which desensitized rapidly upon prolonged application of the neonicotinoids (10 s application). At 1 mM concentration, the maximum currents were −0.46 ± 0.08 for acetamiprid and −0.57 ± 0.09 μA for clothianidin. The mean current amplitudes were 1.71 (P < 0.05, n = 12; Figure 1B) and 1.95 (P < 0.05, n = 12; Figure 1B) fold lower than for ACh. The amplitudes of the clothianidin and acetamiprid‐elicited currents were concentration‐dependent with EC50 values (in mM) of 0.16 ± 0.06 (ACh), 0.74 ± 0.07 (clothianidin) and 0.73 ± 0.06 (acetamiprid). Interestingly, thiamethoxam, which is metabolized to clothianidin only in insects and plants (Nauen et al., 2003; Benzidane et al., 2010), had no agonist effect (Figure 1B, C). Clothianidin and acetamiprid appeared as activators of α7 nAChRs (Figure 1B, C). The dose–response curves were fitted with the Hill equation and the data are shown in Table 1. Thus, despite the close similarity of these three compounds, they differed in their action on rat neuronal α7 nAChRs.

Figure 1.

Figure 1

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Effects of ACh, clothianidin (CLO), acetamiprid (ACE) and thiamethoxam (TMX) on rat α7 neuronal nAChRs. (A) Chemical structure of ACh and the neonicotinoid insecticides studied (clothianidin, acetamiprid, thiamethoxam. (B) Currents induced by bath application of 1 mM ACh, clothianidin and acetamiprid. As shown, 1 mM thiamethoxam induced no current, at any concentration. Each bar indicates when the compound is added. (C) Dose–response curves recorded for control (ACh) and for clothianidin, acetamiprid or thiamethoxam. Responses are normalized to the responses induced by 1 mM ACh and fitted to the Hill equation (see Methods section). Each point plotted in the concentration–response curves represents mean ± SEM of oocytes from 12 different frogs. For each experimental condition, 180 oocytes per batch were recorded.

Table 1.

I max, pEC50 and Hill coefficient values for ACh, clothianidin, acetamiprid and thiamethoxam on rat neuronal α7 nAChRs

I max (μA) pEC50 Hill
ACh 1.0 3.80 ± 0.08 1.192 ± 0.21
Clothianidin 0.57 ± 0.06 3.13 ± 0.05 1.559 ± 0.35
Acetamiprid 0.46 ± 0.09 3.13 ± 0.08 1.435 ± 0.45
Thiamethoxam 0 ND ND
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The values shown (means ± SEM) were determined from the concentration–response curves, generated from oocytes from 12 different frogs. The maximum currents (I max) are shown as the ratio of the maximum response to that for 1 mM Ach (set to unity). ND: not determined.

In the second set of experiments, we studied the modulatory effect of low concentrations of the three neonicotinoid insecticides on ACh‐induced currents in oocytes expressing α7 nAChRs. Coapplication (without pretreatment) of clothianidin or acetamiprid (both 10 μM) with 100 μM ACh (which is close to its EC50) did not change ACh‐evoked current amplitudes (Figure 2A, B). However, pretreatment of the oocytes for 4 min, with the same concentration of clothianidin or acetamiprid (10 μM) before coapplication (10 s) of 100 μM ACh significantly increased ACh‐induced current amplitudes (Figure 2C, D), almost two‐fold. In contrast, no significant effect was found when the oocytes were pretreated with a low concentration of ACh (10 μM) before the coapplication of clothianidin or acetamiprid at the higher concentration (100 μM) (Figure 3A, B). To confirm that pretreatment with 10 μM ACh did not induce desensitization of the receptors or any modulatory effect of the neonicotinoids, we measured responses to 100 μM ACh after pretreatment with 10 μM ACh, and found no effect of this pretreatment on the current induced by the higher concentration of ACh (Figure 3C). We then generated concentration–response curves to ACh after pretreament of the oocytes with clothianidin or acetamiprid (10 μM). As shown in Figure 4A, the concentration–response curves were left‐shifted when compared to the curve obtained with ACh pretreatment. The resulting EC50 values were 0.47 ± 0.09 and 0.89 ± 0.2 mM for the binary combinations ACh/clothianidin and ACh/acetamiprid respectively. The reproducibility of the currents over time was also measured using repeated applications (10 s application at 5 min intervals) of the compounds on the same oocyte. These responses were stable and reproducible, and the potentiation of the ACh‐induced current was maintained, for either neonicotinoid compound, for at least 30 min (Figure 4B).

Figure 2.

Figure 2

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Clothianidin and acetamiprid enhance ACh‐induced current amplitudes of the rat α7 neuronal nAChR. (A and B) coapplication without pretreatment. In the left, currents induced by 100 μM ACh. In the right, coapplication of 10 μM clothianidin (CLO) or acetamiprid (ACE) with 100 μM ACh induces neither a significant increase nor decrease of ACh‐evoked currents. (C and D) Pretreatment with 10 μM clothianidin and acetamiprid. In the left, currents induced by 100 μM ACh. In the middle, effects of 10 μM clothianidin and 10 μM acetamiprid. Clothianidin and acetamiprid alone did not induce currents but, used as a preapplication, strongly increased the subsequent response to ACh (on the right). Each bar in the current indicates when the compound is added. Histograms under the currents illustrate the increase of ACh‐induced current amplitudes after pretreatment with 10 μM clothianidin (C) and acetamiprid (D). Each histogram represents mean ± SEM of oocytes from 12 frogs. For each experimental condition, 120 oocytes per batch were recorded. *P < 0.05, signficantly different as indicated.

Figure 3.

Figure 3

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Responses evoked by 100 μM clothianidin or acetamiprid when oocytes are pretreated with 10 μM ACh. (A and B) Cells are pretreated with 10 μM ACh. In the left, application of 10 μM ACh did not induce any currents. In the middle, responses evoked by 100 μM clothianidin (CLO; upper traces) and 100 μM acetamiprid (ACE; lower traces). In the right, oocytes are pretreated with 10 μM ACh. Exposure to 10 μM ACh alone did not induce currents nor did it affect the clothianidin‐ or acetamiprid‐evoked currents. (C) To confirm that 10 μM ACh does not desensitize the rat α7 neuronal nAChR, we measured ACh‐evoked currents after 4 min pretreatment with 10 μM ACh. In this condition, pretreatment with 10 μM ACh has no effect on the responses evoked by 100 μM ACh. Each bar indicates when the compound is added.

Figure 4.

Figure 4

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Dose–response and time course effects of 100 μM clothianidin and acetamiprid coapplied with 100 μM ACh on oocytes expressing α7 nAChRs. (A) Dose–response relationships of 100 μM ACh alone and coapplied with 10 μM clothianidin (CLO) and 10 μM acetamiprid (ACE). Each point represents mean ± SEM of the mean (n = 12). (B) Time course of ACh of 100 μM ACh (control condition) with 10 μM clothianidin and 10 μM acetamiprid. ACh, clothianidin and acetamiprid are coapplied for 5 s in bath solution, every 5 min. Each point represents mean ± SEM of oocytes from 10 frogs. In each experimental condition, 120 oocytes per batch were recorded. Currents are normalized with 100 μM ACh using the same oocyte. *P < 0.05, signficantly different as indicated.

Thiamethoxam, a poor agonist of nAChRs, inhibits ACh‐ and nicotine‐induced current amplitudes

Based on the previous set of results, thiamethoxam was not able to activate α7 nAChRs. Nevertheless, a low concentration of thiamethoxam (10 μM) decreased the ACh‐induced current amplitudes when it was coapplied with 100 μM ACh (Figure 5A). A similar decrease was observed when the oocytes were pretreated with 10 μM thiamethoxam before coapplication of 100 μM ACh (Figure 5B). In the next set of experiments, thiamethoxam was used as a pretreatment in order to compare its effects with those of clothianidin and acetamiprid. Thus, using the single effective dose of ACh (100 μM) and pretreatment with 10 μM thiamethoxam, the ACh‐activated current was significantly reduced by 33 ± 4% (n = 10; Figure 5C), compared with currents induced by 100 μM ACh alone. Note that when oocytes were pretreated with 10 μM ACh, there was no effect on the response to 100 μM ACh (Figure 5C). As shown in Figure 5D, the concentration–response curve to ACh was right‐shifted by pretreatment with thiamethoxam (10 μM) (Figure 5D) and the effects of different pretreatment concentrations of thiamethoxam (Figure 5E) revealed only a partial inhibition of the ACh‐induced currents, through the α7 nAChRs (Figure 5E). Interestingly, under the same conditions, we demonstrated that 10 μM thiamethoxam completely blocked currents induced after coapplication of 100 μM nicotine (Figure 6A), but pretreatment with 10 μM nicotine had no effect on the response to the high concentration (100 μM) of thiamethoxam (Figure 6B). These data showed that thiamethoxam decreased ACh‐induced and nicotine‐induced current amplitudes, implying an antagonist, rather than agonist, activity of thiamethoxam on rat α7 nAChRs. Moreover, these results were in agreement with the finding that all neonicotinoids showed low binding affinity for mammalian nAChRs but were able to modulate ACh‐induced currents.

Figure 5.

Figure 5

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Modulatory effect of 10 μM thiamethoxam on ACh‐induced currents. (A) Coapplication without pretreatment. Coapplication of 10 μM thiamethoxam (TMX) with 100 μM ACh significantly reduces ACh‐induced current amplitudes. (B) Pretreatment with 10 μM thiamethoxam (4–5 min pretreatment). In the left, current represents response recorded in control condition (100 μM ACh). In the middle, 10 μM thiamethoxam neither induces currents. In the right, 10 μM thiamethoxam strongly decreases ACh‐induced current amplitude. Each bar indicates when the compound is added. (C) Histograms illustrating the decrease of ACh‐induced current amplitudes after pretreatment with 10 μM thiamethoxam. Data are mean ± SEM of oocytes from 10 frogs. *P < 0.05, signficantly different as indicated. Inset, oocytes are pretreated with 10 μM ACh and coapplied with 100 μM thiamethoxam. No effect was found. (D) Dose–response relationship of ACh coapplied with 10 μM thiamethoxam. Each point represents mean ± SEM of oocytes from 10 frogs. (E) Inhibitory curve illustrating the effect of thiamethoxam on ACh responses. Data are plotted as mean ± SEM of oocytes from 10 frogs. In each experimental condition, we used 120 oocytes per batch.

Figure 6.

Figure 6

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Effect of thiamethoxam on nicotine‐induced currents. (A) In the left, current induced by 100 μM nicotine. In the middle, 10 μM did not induce current. In the right, inward currents induced by 100 μM nicotine are completely blocked when oocytes are pretreated with 10 μM thiamethoxam. (B) 10 μM nicotine (left) or thiamethoxam (middle) did not induce currents. Pretreatment of oocytes with 10 μM nicotine did not enhance thiamethoxam‐induced currents (right). Each bar indicates when the compound is added.

Combined applications of clothianidin, acetamiprid and thiamethoxam differently modulate ACh‐induced current amplitudes

The development of insect resistance against pesticides and whole‐field applications has led to the use of mixtures of insecticides (Willmott et al., 2013). As a result, Wang et al., (2017) proposed that the reported negative effect of pesticides could derive from the combination of pesticides. We therefore next investigated the modulatory effect of the three neonicotinoids when used in combination, at the same concentration (10 μM). Combined application of thiamethoxam with clothianidin decreased the peak amplitude of ACh‐induced current by about 50% (n = 12; Figure 7A). Further, combined application of thiamethoxam with acetamiprid strongly reduced ACh currents, inducing about 70% inhibition of the peak ACh current amplitude (n = 12; Figure 7B). Interestingly, we found that the combination of clothianidin and acetamiprid also significantly reduced ACh currents to 47 ± 20% (n = 12; Figure 7C). This finding was unexpected because these neonicotinoids, used separately, had shown agonist effects, as shown above (Figure 1B, C). We therefore tested the concentration‐dependence of this finding, measuring the response to 100 μM ACh in the presence of 10 μM clothianidin and increasing the concentration of acetamiprid. As shown in Figure 7D, pretreatment with 10 μM clothianidin and increasing acetamiprid concentrations antagonized the currents induced by 100 μM ACh. A similar reduction of ACh‐induced currents was found using a combination of the three neonicotinoids, thiamethoxam, clothianidin and acetamiprid, all at 10 μM, (n = 10; Figure 8). Calculations using the CI equation (Zhao et al., 2014; Foucquier and Guedj, 2015) suggested that all neonicotinoid mixtures were antagonistic. The CIs were 2.1, 1.9, 1.2 and 2.4 for the combinations acetamiprid/clothianidin, thiamethoxam /clothianidin, thiamethoxam/acetamiprid and thiamethoxam /acetamiprid /clothianidin respectively.

Figure 7.

Figure 7

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Decrease of ACh‐induced currents after combined application of neonicotinoids. In each condition, in the left, current induced by 100 μM ACh; in the middle, combination of two neonicotinoids tested at 10 μM concentration; and in the right, pretreatment with mixture of neonicotinoids. Pretreatment with 10 μM clothianidin (CLO) (A) and 10 μM acetamiprid (ACE) (A) with 10 μM thiamethoxam (TMX) significantly reduces currents induced by 100 μM ACh. Similar reduction is found with combined application of 10 μM clothianidin with 10 μM acetamiprid (C). Histograms in the right of the recording currents show the decrease of ACh‐induced current amplitudes after pretreatment. In each histogram, data are mean ± SEM of oocytes from 12 frogs. *P < 0.05, signficantly different as indicated. (D) Concentration–inhibition relationship illustrating the inhibition of ACh currents (100 μM ACh) induced by pretreatment with 10 μM clothianidin with different acetamiprid concentrations. Data are plotted as mean ± SEM of oocytes from 10 frogs. In each experimental condition, we used 120 oocytes per batch.

Figure 8.

Figure 8

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Effect of the combination of the three neonicotinoids on ACh‐induced currents. Each neonicotinoid (clothianidin, CLO: acetamiprid, ACE: thiamethoxam, TMX), is used at 10 μM and ACh at 100 μM. ACh‐induced currents are reduced after pretreatment. Bars in the Figure indicate when compound is added. Current are normalized with 100 μM ACh using the same oocyte. Histograms showing the decrease of ACh‐induced current amplitudes. Each histogram represents mean ± SEM of oocytes from 10 frogs. For each experimental condition, we used 120 oocytes per batch. *P < 0.05, signficantly different as indicated.

Although the inhibition observed in the combinations containing thiamethoxam could be explained by the antagonist activity of thiamethoxam, we cannot exclude the possibility that acetamiprid and clothianidin, in a complex mechanism, could act as negative modulators of the α7 nAChRs. Because all combinations of the three neonicotinoids were antagonistic, whereas each neonicotinoid used singly had clearly differing effects, we analysed further the currents induced by combined application of the higher concentrations of the neonicotinoids, after pretreatment with 10 μM ACh. As shown in Figure 9, currents induced by the combined application of thiamethoxam with acetamiprid (both 100 μM) were strongly enhanced after the pretreatment with ACh (n = 10; Figure 9A). Current amplitudes were similarly increased when 100 μM thiamethoxam was combined with 100 μM clothianidin (n = 10; Figure 9B), and by the combination of all three neonicotinoids (n = 10; Figure 9C)]. Note that pretreatment with 10 μM ACh did not change current amplitudes induced by the combined application of 100 μM clothianidin and acetamiprid (Figure 9D), suggesting that thiamethoxam could change the interactions between ACh and neonicotinoid insecticides.

Figure 9.

Figure 9

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Effect of ACh on neonicotinoid‐induced currents. In the present condition, oocytes are pretreated with 10 μM ACh before coapplication with 100 μM neonicotinoids. Pretreatment with 10 μM ACh significantly increases acetamiprid (ACE)/ thiamethoxam (TMX) (A), clothianidin (CLO)/ thiamethoxam (B) and clothianidin/acetamiprid/ thiamethoxam ‐induced current amplitudes (C). Each histogram under the recording currents represents mean ± SEM of oocytes from 10 frogs. In each experimental condition, we used 180 oocytes per batch. *P < 0.05, signficantly different as indicated; NS, not significant. (D) Pretreatment with 10 μM ACh has no effect on current amplitude after combined application of both 100 μM clothianidin and ACE. Each bar in the Figure indicates when the compound is added.

Discussion

Clothianidin and acetamiprid act as partial agonists of α7 nAChRs

Neonicotinoid insecticides are considered as agonists of neuronal nAChRs acting selectively on insect nAChR subtypes (Tomizawa and Casida, 2003; Casida and Quistad, 2004). This assumption is based in part in the study of the commercially used neonicotinoid, imidacloprid. This neonicotinoid did not activate the mammalian α4β2 nAChR subtype expressed in Xenopus oocytes at doses up to 100 μM, but induced a weak activation of the α7 nAChRs (Yamamoto et al., 1998). But responses to imidacloprid were further enhanced in the presence of the E219P mutation in loop C of the α4 subunit (Toshima et al., 2009). In this study, we have demonstrated that clothianidin and acetamiprid were partial agonists of mammalian neuronal α7 nAChRs while thiamethoxam exhibited no agonist activity. Our data are in agreement with studies from Li et al. (2011), demonstrating that clothianidin is a weak agonist of α4β2 receptors with peak current amplitudes of 1–4% of the responses to 1 mM ACh. Interestingly, we found that clothianidin and acetamiprid strongly enhanced ACh‐induced current amplitudes after pretreatment. The effects of clothianidin and acetamiprid were similar to those of ivermectin, a semisynthetic analogue of the natural compound avermectin. Ivermectin acts as a positive allosteric modulator of mammalian α7 nAChRs (Krause et al., 1998; Chatzidaki and Millar, 2015), increasing ACh‐induced currents when it is used as a pretreatment (Krause et al., 1998). In addition, we found that thiamethoxam transiently inhibited ACh‐induced currents, as previously demonstrated with imidacloprid on human α4β2 receptors (Li et al., 2011). Moreover, we demonstrated that thiamethoxam completely blocked nicotine‐induced current amplitudes demonstrating that it acted as an antagonist of α7 nAChRs. Other studies, using imidacloprid, clothianidin and thiacloprid, have already shown that imidacloprid and clothianidin potentiated ACh responses, but thiacloprid attenuated the ACh‐evoked responses in a concentration‐dependent manner when coapplied with ACh (Toshima et al., 2008). These effects were explained by an equilibrium two‐site receptor occupation model in which sequential occupation of the two agonist binding sites by two different agonists greatly enhanced the probability of opening the nAChR channel. If the potentiation induced by clothianidin and acetamiprid could be explained by a similar mechanism, such an explanation would not apply to the effects of combinations of neonicotinoids which include thiamethoxam. Nevertheless, the agonist effect of the second‐generation neonicotinoids such as clothianidin and acetamiprid correlates well with the proposition that these compounds have effects which differ from those of imidacloprid.

Mixtures of neonicotinoids differ in their modulation of ACh‐induced current amplitudes

Eco‐toxicological risk assessments and monitoring of agricultural use of pesticides demonstrate that insecticides are rarely present alone. Indeed, complex mixtures of insecticides could be found in the environment due to adverse weather events, such as storm water run‐off or to spray drift during application. Thus, individuals would be exposed to mixtures of environmental pesticides rather than to one pesticide. In addition, low concentrations of pesticide mixtures can cause significant damage, similar to higher concentrations of individual pesticides (Sultana Shaik et al., 2016). Here, we found that clothianidin, acetamiprid and thiamethoxam, at the same low concentration (10μM), differently modulated ACh‐induced current amplitudes. All combinations using binary or the combination of the three neonicotinoids reduced ACh‐induced current amplitudes, suggesting that when the three neonicotinoids were used in combination, they reduced ACh‐evoked currents. Note that a strong modulatory effect was found using the combination of acetamiprid with thiamethoxam. We proposed that these compounds synergize the effect of ACh. Moreover, we suggested that synergism leading to the reduction of ACh currents could be due to the presence of thiamethoxam. In the case of binary combination between clothianidin and acetamiprid, the reduction was associated with clothianidin rather than acetamiprid, because clothianidin was known to be a metabolite of thiamethoxam. Future studies will reveal to what extent our findings could affect cholinergic neurotransmission in vivo.

Agonist effect of clothianidin and acetamiprid associated with their chemical structure

Clothianidin and thiamethoxam possess a heterocyclic aromatic moieties coupled to a cyclic or an acyclic N‐nitroimine moiety, whereas acetamiprid possess an N‐cyanoimine moiety. It appears that the chemical structures of clothianidin and acetamiprid give rise to the possibility to bind as full agonists of insect neuronal nAChRs (Tan et al., 2007; Thany, 2009) and partial agonists of mammalian neuronal nAChRs (Li et al., 2011), compared with thiamethoxam. Thiamethoxam is a second‐generation commercial neonicotinoid that belongs to the thianicotinyl sub‐class (Maienfisch et al., 2001). As demonstrated in the present study and several other publications, clothianidin and thiamethoxam did not have a similar mode of action on insect nAChR subtypes, even though thiamethoxam is metabolized to clothianidin (Nauen et al., 2003; Ford and Casida, 2006; Benzidane et al., 2010). Thus, although the effects of thiamethoxam are still a matter of debate, our results would support the hypothesis that it can act on mammalian nAChRs as an antagonist, which would be in agreement with previous studies demonstrating that it was able to induce its own toxic effect (Wellmann et al., 2004). Moreover, we found that thiamethoxam partly blocked ACh‐induced currents and completely blocked nicotine currents. Based on our studies and considering the chemical structure of thiamethoxam, this selective action could be explained by the nature of the α/α7 interfaces in the nAChR where the α subunits provide five potential binding sites for agonists (Papke, 2014; Dani, 2015) but with low affinity for nicotine (Schapira et al., 2002; Dani, 2015). Therefore, it is reasonable to propose that thiamethoxam would be a more potent antagonist of nicotine‐induced currents, than those induced by ACh.

Working hypothesis: is it possible to suggest that neonicotinoid insecticides could act as allosteric modulators of neuronal nAChRs?

Neonicotinoids negatively affect pollinating insects, in several ways, because they are present in the nectar and pollen of plants after application (Bonmatin et al., 2003; Henry et al., 2012; Rondeau et al., 2014; Sanchez‐Bayo, 2014; Simmons and Angelini, 2017). Due to their potential toxicity to honey bees, imidacloprid, thiamethoxam and clothianidin were extensively examined by the European Commission, in terms of the effects of the currently authorized uses of these substances as seed treatment and granules (see regulation no. 485/2013). Electrophysiological studies demonstrated that their toxic effects on bees were associated with their agonist activity on insect neuronal nAChRs (Matsuda et al., 2001; Thany et al., 2007). However, the actions of neonicotinoids on mammalian nAChRs remain the subject of much debate worldwide, in terms of their possible adverse effects on human health (Marfo et al., 2015; Sheets et al., 2016; Seltenrich, 2017). One relevant finding is that they are poor agonists of mammalian nAChRs (Tomizawa and Casida, 2003). Although the main focus of the present study was not allosteric mechanisms, our results have led us to suggest that neonicotinoids could act as allosteric modulators of mammalian neuronal nAChRs. From our data, clothianidin and acetamiprid could be positive allosteric modulators and thiamethoxam could be a negative allosteric modulator. Our hypothesis would be supported by the finding that clothianidin or acetamiprid enhanced ACh‐evoked currents after pretreatment. However, as previously demonstrated using the combination of several pesticides (Yi et al., 2012; Taillebois and Thany, 2016), our results could be due to additive, synergistic or antagonist effects of the three neonicotinoids with ACh, on the α7 nAChRs. Our data also suggest that the three neonicotinoid insecticides have complex effects on mammalian neuronal nAChRs. Thus, in the light of previous data, it seems reasonable to suggest that these neonicotinoids each differ in their ability to alter the functional properties and binding of ACh to α7 nAChRs. Nevertheless, for all three compounds studied here, our data suggest that there is a strong interaction between the neonicotinoids and ACh on rat α7 neuronal nAChRs, which leads to a modulation of ACh‐induced currents.

Author contributions

S.H.T. designed the experiments; A.C. and C.M. performed the experiments; A.C. and S.H.T. analysed the data and wrote the manuscript.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This http://onlinelibrary.wiley.com/doi/10.1111/bph.13405/abstract acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

A.C. received a PhD grant from the Region Centre Val de Loire. We gratefully acknowledge Roger Papke, Clare Stokes (University of Florida) and Millet Treinin (Hebrew University of Jerusalem) for the provision of α7 cDNA and RIC3.

Cartereau, A. , Martin, C. , and Thany, S. H. (2018) Neonicotinoid insecticides differently modulate acetycholine‐induced currents on mammalian α7 nicotinic acetylcholine receptors. British Journal of Pharmacology, 175: 1987–1998. doi: 10.1111/bph.14018.

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