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

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.

Figure 1

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.

Figure 2

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

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

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.

Figure 5

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

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.

Figure 7

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

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.

Figure 9

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.