Desensitization of nicotinic acetylcholine receptors in ...

Insect Biochemistry and Molecular Biology 41 (2011) 872e880

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Desensitization of nicotinic acetylcholine receptors in central nervous system neurons of the stick insect (Carausius morosus) by imidacloprid and sulfoximine insecticides Eugênio E. Oliveiraa, b, *, Sabine Schleichera, b, Ansgar Büschgesa, Joachim Schmidta, Peter Kloppenburga, b, Vincent L. Salgadoc, ** a

Institute for Zoology, Cologne Biocenter, University of Cologne, 50674 Cologne, Germany Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Germany c BASF Corporation, 26 Davis Drive, Research Triangle Park, NC 27709, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 February 2011 Received in revised form 9 August 2011 Accepted 9 August 2011

Imidacloprid, sulfoxaflor and two experimental sulfoximine insecticides caused generally depressive symptoms in stick insects, characterized by stillness and weakness, while also variably inducing postural changes such as persistent ovipositor opening, leg flexion or extension and abdomen bending that could indicate excitation of certain neural circuits. We examined the same compounds on nicotinic acetylcholine receptors in stick insect neurons, which have previously been shown to desensitize in the presence of ACh. Brief U-tube application of 104 M solutions of insecticides for 1 s evoked currents that were much smaller than ACh-evoked currents, and depressed subsequent ACh-evoked currents for several minutes, indicating that the compounds are low-efficacy partial agonists that potently desensitize the receptors. Much lower concentrations of insecticides applied in the bath for longer periods did not activate currents, but inhibited ACh-evoked currents via desensitization of the receptors. Previously described fast- and slowly-desensitizing nACh currents, IACh1 and IACh2 respectively, were each found to consist of two components with differing sensitivities to the insecticides. Imidacloprid applied in the bath desensitized high-sensitivity components, IACh1H and IACh2H with IC50s of 0.18 and 0.13 pM, respectively. It desensitized the low-sensitivity slowly desensitizing component, IACh2L, with an IC50 of 2.6 nM, while a component of the fast-desensitizing current, IACh1L, was least sensitive, with an IC50 of 81 nM IACh1L appeared to be insensitive to the three sulfoximines tested, whereas all three sulfoximines potently desensitized IACh1H and both slowly desensitizing components, with IC50s between 2 and 7 nM. We conclude that selective desensitization of certain nAChR subtypes can account for the insecticidal actions of imidacloprid and sulfoximines in stick insects. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Acetylcholine Nicotinic acetylcholine receptors Insecticides Sulfoximines Neonicotinoids

1. Introduction Insecticides targeting receptors are excellent tools for the characterization of those receptors. The development of the receptor concept itself originated from studies by Schmiedeberg and Koppe (1869) on the physiological effects of the natural insecticide

* Corresponding author. Present address: Department of Entomology, Genetics and Neuroscience Programs, Michigan State University, 438 Giltner Hall, East Lansing, MI 48824, USA. Tel.: þ1 517 353 5571; fax: þ1 517 353 4354. ** Corresponding author. Tel.: þ1 919 547 2244; fax: þ1 919 547 2450. E-mail addresses: [email protected] (E. E. Oliveira), [email protected] (V.L. Salgado). 0965-1748/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2011.08.001

muscarine from fly agaric (Amanita muscaria) and the seminal work by Langley (1905) on the natural insecticide nicotine. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channel receptor complexes that mediate fast cholinergic synaptic transmission. They are among the most thoroughly studied molecules in nervous systems. In vertebrates, neuronal nAChRs consist of pentameric ligand-gated cation channels formed by the assembly of multiple a (a2ea10) and b (b2eb4) subunits, different combinations of which result in distinct receptor subtypes with distinctive pharmacological profiles (Tomizawa and Casida, 2001, 2003). In insects, more than 10 nAChR genes have been cloned, and post-translational modification of subunit mRNAs has been demonstrated (Grauso et al., 2002; Lansdell and Millar, 2002; Matsuda et al., 2005; Millar and Gotti, 2009; Sattelle et al., 2005), suggesting the existence of many nAChR subtypes.

E.E. Oliveira et al. / Insect Biochemistry and Molecular Biology 41 (2011) 872e880

Neonicotinoids (Ihara et al., 2006; Jeschke and Nauen, 2008; Matsuda et al., 2009; Tomizawa and Casida, 2003), spinosyns (Orr et al., 2009; Salgado, 1998a; Salgado and Saar, 2004; Salgado et al., 1998b) and nereistoxin analogs (Delpech et al., 2003; Lee et al., 2003), are insecticide classes known to selectively target insect nAChRs, each acting via a different mechanism. Neonicotinoids, agonists that mimic the action of acetylcholine, enjoy widespread use against a broad spectrum of sucking and certain chewing insect pests (Jeschke and Nauen, 2008; Matsuda et al., 2001). Imidacloprid is the top-selling insecticide worldwide, and another neonicotinoid, thiamethoxam, is second (PhillipsMcDougall, 2010). Nicotine itself is the original nicotinic insecticide, but is being phased out due to its high mammalian toxicity (EPA, 2008, 2009). Recently, a new class of nicotinic agonists known as sulfoximines has been reported (Zhu et al., 2006). Sulfoxaflor is a sulfoximine that is being developed by Dow AgroSciences. Sulfoximines have been reported to cause excitatory symptoms in aphids and to activate nicotinic receptors composed of Drosophila a2 and b2 subunits heterologously expressed in Xenopus oocytes, and to displace [3H]-imidacloprid from membranes of the aphid Myzus persicae. However, while sulfoxaflor was comparable to imidacloprid in insecticidal potency, it’s affinity in displacing imidacloprid was at least 50-fold lower (Watson et al., 2011; Zhu et al., 2010), indicating that the action of the sulfoximines is not adequately understood. We used the stick insect Carausius morosus for the present study. Locust (e.g., Burrows, 1996), cockroach (Ritzmann and Büschges, 2007) and stick insects have been used extensively as model systems for the study of insect motor behavior, particularly walking. Today, the most complete picture on neural network action underlying insect walking behavior exists for the stick insect (e.g., recent summaries in Büschges et al., 2008; Orlovsky, 1999; Ritzmann and Büschges, 2007). Also, in the stick insect the role of cholinergic receptors in neurons of the locomotor network has been studied (Büschges, 1998; Oliveira et al., 2010; Westmark et al., 2009). While both desensitizing and nondesensitizing nicotinic receptors are found in cockroach neurons (Salgado and Saar, 2004), only desensitizing nAChRs have been observed in stick insect neurons (Oliveira et al., 2010). The purpose of the present study was to characterize the actions of imidacloprid and sulfoxaflor on these two desensitizing nAChRs, in order to better understand the basis for the insecticidal activity of these compounds and to characterize them for use as potential tools for studying the roles of the different receptor subtypes in the insect nervous system. 2. Materials and methods Patch-clamp experiments were carried out on isolated unindentified stick insect thoracic neurons, as previously described (Oliveira et al., 2010; Westmark et al., 2009). Briefly, three thoracic ganglia were removed and immersed in cold, sterile ‘normal’ extracellular saline containing (in mM): 180 NaCl; 4 KCl; 5 CaCl; 1 MgCl 2; 10 HEPES and 48 sucrose, adjusted to pH 7.2 with NaOH and 430 mOsm with sucrose, where the ganglionic sheath was manually removed. To soften the exposed perineurium, the ganglia were next treated for 10 min with a mixture of collagenase (250 units ml1, C0130, SigmaeAldrich) and trypsin (8550 units ml1, T8003, SigmaeAldrich) in Hanks’ Ca2þ- and Mg2þ-free buffered salt solution (14170-070, GIBCO, Invitrogen, Karlsruhe, Germany), containing (in mM): 10 HEPES and 135 sucrose, adjusted to pH 7.2 with NaOH. This and all subsequent isolation steps were carried out at room temperature. Next, the ganglia were transferred to normal extracellular solution and the perineurium was manually removed. To facilitate dissociation of

873

the cell bodies, the ganglia were enzyme-treated for a second time (20 min). After rinsing at least five times with normal extracellular saline, the ganglia were dissociated by gentle trituration through a series of pipettes of decreasing tip diameter. The neurons of one ganglion were plated in 1e2 culture dishes. Dishes with glass bottoms were custom-made as described by Oliveira et al. (2010). The dishes were sterilized with UV radiation for 2 h and the glass bottom was coated with concanavalin A (0.7 mg ml1 dissolved in H2O, C-2010, SigmaeAldrich, Taufkirchen, Germany). Neurons were allowed to settle and adhere for at least 1 h, and were used for electrophysiological experiments within 8 h. For recording, cells were visualized with an inverted microscope (IX71, Olympus, Hamburg, Germany) using a 40 water immersion objective (UAPO 40, 1.15 numerical aperture, 0.25 mm working distance, Olympus). Whole-cell recordings were performed at 24 C, following the methods described by Hamill et al. (1981). Electrodes with tip resistances between 3 and 5 MU were fashioned from borosilicate glass (0.86 mm inner diameter, 1.5 mm outer diamenter, GB150-8P, Science Products, Hofheim, Germany) with a temperature-controlled pipette puller (PIP5, HEKA-Elektronik, Lambrecht, Germany), and filled with a solution containing (in mM): 190 K-aspartate, 10 NaCl, 1CaCl2, 2 MgCl2, 10 Hepes, 10 EGTA, 3 ATP and 3 GTP adjusted to pH 7.2 (with KOH), resulting in an osmolarity of w415 mOsm. During the experiments, if not stated otherwise, the cells were superfused constantly with normal extracellular saline solution. Whole-cell voltage-clamp recordings were made with an EPC9 patch-clamp amplifier (HEKA-Elektronik) that was controlled by the program Pulse (version 8.63, HEKA-Elektronik) running under Windows. Electrophysiological data were low-pass filtered at 2 kHz with a 4-pole Bessel-Filter and sampled at intervals of 100 ms (10 kHz). Compensation of offset potential and capacitance were performed using the ‘automatic mode’ of the EPC9 amplifier. The liquid junction potential between intracellular and extracellular solutions of 15.8 mV (calculated with Patcher’s-Power-Tools plug-in from http://www.mpibpc.gwdg.de/abteilung/140/software/index. html for Igor Pro 6 Wavemetrics, Portland, Oregon, USA]) was also compensated. Voltage errors due to series resistance (RS) were minimized using the RS-compensation of the EPC9. RS was compensated between 30% and 70% with a time constant (s) of 2 ms. The cells were held at 60 mV, which is near their normal resting potential (Schmidt et al., 2001). The neurons were continuously superfused with normal extracellular saline (0.5 ml min1) through a teflon tube (483 mm inner diameter) placed 600 mm from the cell. ACh was applied for 1 s with a U-tube system as described previously (Oliveira et al., 2010; Westmark et al., 2009; Wu et al., 2004; Zhao et al., 2003). Acetylcholine (ACh, A2661) and imidacloprid (IMI, 37894) were purchased from SigmaeAldrich (Taufkirchen, Germany). The sulfoximines (Fig. 1) were synthesized according to published procedures (Loso et al., 2007; Zhu et al., 2006). Imidacloprid and the sulfoximines were dissolved in and serially diluted in dimethyl sulfoxide (DMSO, D8418, SigmaeAldrich) before being suspended in saline with a maximum final DMSO concentration of 0.1% (vol/ vol). DMSO alone in its final concentration had no effect on neurons. All other ligands were dissolved in and diluted in normal extracellular saline. The amplitudes of ACh-induced currents (IACh1: peak current. IACh2: current amplitude averaged between 950 ms and 1000 ms of ACh application) were determined as in Oliveira et al. (2010). To determine concentrationeresponse relations for desensitization of ACh-induced currents by insecticides, the current evoked by pulses of ACh at 1 min intervals was followed at each insecticide concentration until a steady-state effect was achieved. For sulfoximines,

874


A

imidacloprid

sulfoxaflor

O N

N

For observation of poisoning symptoms, compounds dissolved in 1 ml of DMSO were injected dorsolaterally into the abdomen of adult insects with a mean weight of 0.9 g, which were all females in this parthenogenetic species, using a 10 ml syringe fitted with a 30 gauge needle. Injected animals, 3e5 at each dose, were offered blackberry leaves and sprayed with water to maintain moisture levels.

N

O N

N

N

S O

Cl

N

F 3C

N

3. Results

sulfoximine 1 O

sulfoximine 2

N

N

N N

S S O Cl

B

N

Cl

N

ACh

100 pA 1 s Fig. 1. A. The structures of imidacloprid, sulfoxaflor and sulfoximines 1 and 2. B. Acetylcholine-induced current (IACh) at a holding potential of 60 mV. ACh was applied for 1 s (104 M), eliciting a fast-desensitizing (IACh1) and a slow-desensitizing current component (IACh2), as recently described.

concentrationeresponse relations for each cell were fit with the Hill equation for a single binding site:

I ¼ Imax

1 ½ligandnH 1þ knH

(1)

where Imax is the current amplitude in the absence of ligand, k is the IC50 (ligand concentration that reduced the current by 50%), and nH is the Hill coefficient. Data were scaled as a fraction of the calculated maximal current and re-fit. For imidacloprid concentrationeresponse relations, a two-site Hill equation was used:

I ¼ Imax

fH fL þ ½imidacloprid ½imidacloprid 1þ 1þ kH kL

(2)

where fH is the fraction of total sites that have high sensitivity to imidacloprid and kH is their IC50, while fL and kL are the corresponding values for the low-sensitivity site. fH þ fL ¼ 1. Hill coefficients for both fractions were not included and were assumed to be 1. Fitting of imidacloprid concentrationeresponse data was done with global nonlinear curve fitting in GraphPad Prism software (GraphPad Software, INC., La Jolla, USA). Electrophysiological data were analyzed with the software Pulse (version 8.63; HEKA), Igor Pro 6 (Wavemetrics, INC., Lake Oswego, USA; including the Patcher’s-Power-Tools plug-in, http://www. mpibpc.gwdg.de/abteilungen/140/software/index.html) and Sigma Stat (Systat Software, INC., San Diego, USA). If not stated otherwise, all calculated IC50 values are expressed as mean followed by their 95% confidence interval (CI) values.

Imidacloprid and the three sulfoximines were all toxic to stick insects by injection. Doses were chosen to enable observation of symptoms at minimally toxic doses, with the minimum number of animals. Table 1 shows the percent of injected animals that were prostrate at times up to 3 days post-injection. None of the compounds caused excitatory symptoms, and while there were some similarities, sulfoximine symptoms were clearly different from imidacloprid symptoms. Imidacloprid-injected animals became still and responded to touch more weakly than normal. The legs became flexed, causing the animals to become prostrate, either lying on their sides, or ventral side down. Other symptoms seen were dorsad bending of the abdomen, persistent opening of the ovipositor, and tarsal flexion or extension, but these were not observed in all insects. Isolated leg tremors sometimes occurred, but were rare. Injected insects were knocked down at all three doses, but were able to stand and walk again within 3 days. At 0.2 mg, partial recovery was evident within 2 h. However, weakness persisted, and at the highest dose (2 mg), the insects eventually fell over again and died by the 11th day. Sulfoximine symptoms began with pronounced dorsad curvature of the abdomen, and persistent full opening of the ovipositor. These symptoms were pronounced in most insects, and persisted for several hours, even after prostration. Tarsal flexion, leg extension and spitting up clear fluid or white foam were also observed. As with imidacloprid, the animals became noticeably stiller, with decreased locomotory movements. Weakness also occurred, but this set in somewhat after the other symptoms, and after the insects were prostrate. Initially, insects were still but responded to touch with normal reflexes; however these responses gradually became weaker. Sulfoximine 1 was only tested at 20 mg per insect. It caused 60% prostration after 2 h, but insects recovered completely within 18 h. Sulfoxaflor and its chloro analog sulfoximine 2, were equally active, causing 60% prostration within 2 h at 2 mg, but insects recovered much more rapidly from sulfoxaflor than from sulfoximine 2. At 6 mg, 100% of insects were prostrate, but while 60% of sulfoxaflor-treated insects recovered, all of the sulfoximine 2-injected insects eventually died.

Table 1 Percent prostration at various times after injection of the indicated doses of imidacloprid and sulfoximines in stick insects with a mean body weight of 0.9 g. Time (h)

0.5 1 2 8 18 22 30 42 51 72

Sulfoxaflor (mg)

Imidacloprid (mg)

Sulfoximine 1(mg)

Sulfoximine 2 (mg)

0.2

0.6

2

2

6

20

20

2

6

20

0 100 80 80 20 20 0 20 60 0

0 100 100 80 100 100 20 0 0 0

100 e e e 100 e e 33 e 0

0 60 60 40 0 0 0 0 0 0

20 100 100 100 100 100 100 40 40 40

0 e 100 100 100 100 100 100 100 100

0 e 66 e 0 0 0 0 0 0

0 e 60 60 60 60 60 60 40 0

0 e 80 100 100 100 100 100 100 100

0 e 100 100 100 100 100 100 100 100


Using the whole-cell voltage-clamp method, we investigated the actions of three sulfoximines (Fig. 1A) on ACh-induced currents (IACh), which consisted of fast- and slow-desensitizing components, IACh1 and IACh2, respectively, with no detectable muscarinic component (see Oliveira et al., 2010, Fig. 1B). In all cells studied, ACh responses were completely desensitized by bath-applied Imidacloprid (up 106 M) without activation of nondesensitizing currents, confirming that all measurable nicotinic receptors in these cells desensitize, as found previously (Oliveira et al., 2010). Proportions of fast- and slow-desensitizing components varied greatly between cells. Imidacloprid, an insecticide known to target

A

saline

AC h

875

insect nAChRs (Ihara et al., 2006; Jeschke and Nauen, 2008; Matsuda et al., 2001; Salgado and Saar, 2004; Tomizawa and Casida, 2003), served as a reference for the sulfoximine actions. In order to determine whether sulfoximines have agonist activity on stick insect nAChRs, 104 M solutions of the compounds were pulsed onto cells using the U-tube. Fig. 2 shows sulfoximineinduced currents in a cell with primarily fast-desensitizing ACh receptors. In this case, all three sulfoximines induced small currents, showing that they have agonist activity. ACh responses were depressed immediately after sulfoximine application, but recovered within 3 min in all cases. Imidacloprid did not induce a current in this cell, but it depressed subsequent ACh responses for up to 3 min, indicating that it desensitized the ACh receptors (Fig. 2E). Another example of insecticide-induced currents is shown in Fig. 3, using a cell with prominent fast- and slow-desensitizing ACh current components. In this case, sulfoxaflor and sulfoximine 1-induced small currents, but sulfoximine 2 did not. Nevertheless,

A B

sulfoxaflor

AC h

B C sulfoximine 1

AC h

C D

sulfoximine 2

ACh

D

E

imidacloprid

ACh

AC h

Fig. 2. Sulfoximine- and imidacloprid-evoked currents. All recordings were performed in the same neuron. Substances were applied at a concentration of 104 M. The holding potential was 60 mV. A. ACh-induced inward current. B., C., D. The three sulfoximines evoked inward currents (left). ACh-evoked currents were reduced immediately after the sulfoximine application, but recovered after >3 min wash (right). E. Imidacloprid did not evoke an obvious current, but the reduced ACh current (ACh*) 3 min after imidacloprid application indicated an imidacloprid effect. The ACh current further recovered after 5 min wash (right).

E

Fig. 3. Sulfoximine- and imidacloprid-evoked currents. All recordings were performed in the same neuron. Substances were applied at a concentration of 104 M. The holding potential was 60 mV. A. ACh-induced inward current. B., C., E. Sulfoxaflor, sulfoximine 1 and imidacloprid evoked inward currents (left). ACh-evoked currents were reduced immediately after the ligand application, but recovered after >2 min wash (right). D. Sulfoximine 2 did not evoke an obvious current, but the reduced ACh current (ACh*) 1.5 min after sulfoximine 2 application indicated a sulfoximine 2 effect. The ACh current recovered after 2.5 min wash (right).

876


A

sulfoximine 2 did reversibly depress subsequent ACh responses (Fig. 3D). In this cell, imidacloprid activated currents with clear fastand slow-desensitizing components. While the above results confirm that the sulfoximines are agonists of stick insect nAChRs and induce small currents when pulse-applied, the desensitizing effects of bath-applied imidacloprid or sulfoximines on currents evoked by 1 s pulses of 104 M ACh were studied in subsequent experiments, to mimic exposure of the receptors to the insecticides in a poisoned insect (Salgado and Saar, 2004). Inhibition of both IACh1 and IACh2 by bath-applied imidacloprid was highly variable, and the concentrationeresponse relations were similar for IACh1 and IACh2 (Fig. 4). Imidacloprid at 1013 M, the lowest concentration tested, inhibited IACh1 and IACh2 by 31 8 and 39 5%, respectively. IACh was completely abolished by 106 M imidacloprid, and it partially recovered with extended washing (w65% recovery after 25 min wash). At 1012 M, imidacloprid inhibited IACh1 and IACh2 more than it did at 1013 M, but the level of inhibition plateaued between 1012 M and 1011 M, indicating that 50e60% of the receptors mediating both the IACh1 and the IACh2 currents are extraordinarily sensitive to imidacloprid, with IC50’s below 1013 M (Fig. 4B). At concentrations between 1010 and 106 M, IACh1 and IACh2 currents were inhibited further by imidacloprid. Together, these findings indicate that IACh1 and IACh2 each

IACh1

1.0

cell 1 cell 2

0.5

0

B

1.0

IACh2

cell 3 cell 4

0.5

0 -14

-12

-10

-8

-6

Log [imidacloprid (M)] -13

control ACh

IMI (10

M)

ACh

-11

IMI (10

M)

ACh

IACh2 200 pA 1s

IACh1 -9

-7

IMI (10 M) ACh

wash

IMI (10 M)

ACh

ACh

1.0

IACh1

IACh2

consists of a high- and a low-sensitivity component, so that we can distinguish a total of four components of the ACh-evoked currents, which we named IACh1H, IACh1L, IACh2H and IACh2L. The high variability in the action of imidacloprid seen in the averaged data of Fig. 4B is proposed to be due to variation in the proportions of high- and lowsensitivity components of IACh1 and IACh2 between cells. The best-fit IC50 values for high (kH)- and low (kL)-sensitivity components of IACh1 and IACh2 currents were determined by global nonlinear curve fitting of the concentrationeresponse relations for each of these currents in five cells with a two-site Hill equation (Equation (2)). Data for each current (IACh1 and IACh2) yielded a family of five curves, one for each cell, that were fit simultaneously, allowing the proportions of high (fH)- and low (fL)-sensitivity components to vary between cells, while the IC50 values were shared among all cells (Fig. 5). The best-fit IC50 values are shown in Table 2 Effects of imidacloprid and sulfoximines on IACh.

0.5

0

Fig. 5. Examples of concentrationeresponse relations in single neurons for desensitization of IACh1 (A) and IACh2 (B) by imidacloprid. The continuous curves were drawn according to a two-site binding isotherm with the IC50 values in Table 1 and the fraction of the high-sensitivity components from Table 2, which were derived from global nonlinear curve fitting with the two-site Hill equation, as described in the text.

n=5 -12

n=5 -8 -10 -6 Log [IMI (M)]

-12

-10 -8 -6 Log [IMI (M)]

Fig. 4. Imidacloprid-induced desensitization of IACh1 and IACh2. A. IACh was induced by application of 104 M ACh. Imidacloprid was bath-applied at the indicated concentrations, at a holding potential of 60 mV. B. Concentrationeresponse relations for desensitization of IACh1 and IACh2 by imidacloprid. Current amplitudes were scaled as a fraction of IACh1 and IACh2 evoked by 104 M ACh in the absence of imidacloprid. Although the level of inhibition was somewhat variable, it is obvious that IACh1 and IACh2 both have components with differential sensitivity to imidacloprid. The highsensitivity currents (IACh1H and IACh2H) were both inhibited completely by 1012 M imidacloprid, while the low-sensitivity components (IACh1L and IACh2L) were inhibited with IC50 values of 81 109 M (47 109 M 140 109 M, nH ¼ 1.0, n ¼ 5) and 3 109 M (1 109 M 6 109 M, nH ¼ 1.0, n ¼ 5), respectively.

Imidacloprid (n ¼ 5)

IC50 (M)

nH

IACh1H IACh1L IACh2H IACh2L

2 1013 (1 1013e3 1013) 81 109 (47 109 140 109) 1 1013 (0.8 1013e2 1013) 3 109 (1 109e6 109)

1.0 1.0 1.0 1.0

Sulfoximines

IC50 (M)

nH

7 109 (5 10912 109) 2 109 (1 1095 109)

0.8 0.08 0.9 0.15

2 109 (1 1093 109) 3 109 (1 1097 109)

1.0 0.11 0.8 0.10

5 109 (2 10915 109) 3 109 (0.9 1098 109)

0.6 0.10 0.7 0.12

Sulfoxaflor (n ¼ 6) IACh1H IACh2 1 (n ¼ 4) IACh1H IACh2 2 (n ¼ 4) IACh1H IACh2

IC50 is the concentration that inhibits half of the maximal current. 95% confidence intervals of IC50 values are presented in parentheses. nH is the Hill coefficient. nH values are mean SD.


A

Table 3 Fraction of IACh1 and IACh2 that is high affinity (fH) in five cells. Cell

fACh1H

1 2 3 4 5

0.72 0.34 0.25 0.92 0.50

a b

2

a

0.07 0.07 0.11 0.06 0.08

R

fACh2H

0.90 0.97 0.98 0.81 0.89

0.90 0.10 0.45 0.10 0.50 0.16 1 0.20 0.13

-6

control

sulfoxaflor (10 M)

AC h

2

b

R

0.91 0.95 0.92 0.99 0.94

877

IAC ACh2 h2

wash ACh

ACh

IAC ACh1L h1L

100 pA

IACh1

Means the fH value for IACh1. Means the fH value for IACh2.

1 1s

B Table 2 and the proportion of high-sensitivity components of each current (IACh1 and IACh2) in each cell are shown in Table 3. The concentrationeresponse relations for desensitization of IACh1 and IACh2 by imidacloprid in the five cells studied were well fit by the two-site Hill model, with R2 > 0.81 in all cases. The high-sensitivity components of IACh1 and the IACh2 were potently desensitized by imidacloprid, with IC50s of 2 1013 M (1 1013 Me3 1013 M, nH ¼ 1.0, n ¼ 5) and 1 1013 M (0.8 1013 Me2 1013 M, nH ¼ 1.0, n ¼ 5), respectively. The low-sensitivity component of IACh2 was desensitized by imidacloprid with an IC50 of 3 109 M (1 109 Me6 109 M, nH ¼ 1.0, n ¼ 5), while the low-sensitivity component of IACh1 was least sensitive to imidacloprid, with an IC50 of 81 109 M (47 109 Me140 109 M, nH ¼ 1.0, n ¼ 5). The three sulfoximines had clear inhibitory effects on IACh1 and IACh2 (Figs. 6e9). While the sulfoximines inhibited IACh2 completely, they only partially inhibited IACh1. Inhibition of both IACh1 and IACh2 were reversible. For IACh1 the concentrationeresponse relation for the inhibition by sulfoximines plateaued above 106 M, leaving a large portion of IACh1 unblocked (Figs. 6, 7A and 8A, 9A). Because the sulfoximineinsensitive component and the imidacloprid low-sensitivity component IACh1L showed similar desensitization time constants (Imidacloprid (1012 M): 5.1 1.5 ms, n ¼ 5. Sulfoximines (106 M): 5.0 2.1 ms, n ¼ 14; p ¼ 0.48, unpaired t-test), we assumed that they are the same component of IACh1. To determine the concentrationeresponse relation for the sulfoximine-sensitive component (IACh1H; Fig. 7B,C, 8B,C, 9B,C) we subtracted IACh1L from IACh1 for each sulfoximine concentration, as exemplified in Fig. 6. The concentrationeresponse relations for the inhibition of IACh1H were well fit with a single binding site model with different IC50s for each sulfoximine: Sulfoxaflor (Fig. 7C): 7 109 M (5 109 Me12 109 M), nH ¼ 0.8 0.08, n ¼ 6. Sulfoximine 1 (Fig. 8C): 2 109 M (1 109 Me3 109 M), nH ¼ 1.0 0.11; n ¼ 4.

A

B

control AC h

IACh2

sulfoxaflor-sensitive currents sulfoxaflor (10 M) ACh

100 pA

IAC ACh2 h2

IACh1H

1s -8

-7

sulfoxaflor (10 M)

sulfoxaflor (10 M)

ACh

ACh

C 1.0

0.5

IACh1H IACh1 0

IACh2

n=4

n=4

-10 -8 -6 -4 Log [sulfoxaflor (M) ]

-8 -6 -4 -10 Log [sulfoxaflor (M) ]

Fig. 7. Sulfoxaflor-induced desensitization of IACh1 and IACh2. A. Sulfoxaflor (106 M) inhibited IACh2 completely, suggesting that IACh2H and IACh2L are both sensitive. However, IACh1 was only partially inhibited by sulfoxaflor at that concentration, indicating that IACh1 consists of a low (IACh1L) and a high (IACh1H) sensitivity component. The sulfoxaflor effects were reversible (w80% recovery after 30 min wash). B. IACh1H was obtained by subtraction of IACh1L (A, middle trace) from the total current. Sulfoxaflor was bath-applied at the indicated concentrations. C. Concentrationeresponse relations of sulfoxaflor for inhibition of IACh1H and IACh2. The experimental conditions were as described in Fig. 2B. From fits with the Hill equation (Eq. (1)), we estimated an IC50 for IACh2 of 2 109 M (1 109 Me5 109 M), nH ¼ 0.9 0.15 and 7 109 M (5 109 Me12 109 M), nH ¼ 0.8 0.08 for IACh1H.

-6

sulfoxaflor (10 M) AC h

C

ACh

IACh1L 100 pA

IACh1

-9

control ACh

IACh1H

20 ms

Fig. 6. ACh-evoked currents at an expanded time scale to demonstrate the measurement of sulfoximine-sensitive currents, using sulfoxaflor as an example. Current was induced by application of ACh (104 M) at a holding potential of 60 mV before (A) and (B) in the presence of 106 M sulfoxaflor. The residual current is insensitive to sulfoxaflor and is assumed to be the component with low-sensitivity to imidacloprid (IACh1L). C. Subtraction of B from A yields the sulfoximine-sensitive current, composed of IACh1H and IACh2.

878


A

control

-6

sulfoximine 1 (10 M)

ACh

A

control

-9


ACh

ACh

ACh

IACh1L

100 pA

IACh1

wash ACh

ACh

IACh2

IACh1L

IACh2

wash

400 pA

1s

IACh1

B

sulfoximine 1-sensitive currents -9


control ACh

1s

B

sulfoximine 2-sensitive currents

ACh

-9

control


ACh

IACh1H

IACh2

ACh

100 pA

IACh2

200 pA

1s -8

sulfoximine 1 (10 M) ACh

IACh1H

-7


1s -8


ACh

AC h

C

-7

sulfoximine 2 (10 M) AC h

1.0

C 1.0 0.5

IACh1H IACh1 0 n=4

-10 -8 -6 -4 Log [sulfoximine 1 (M)]

IACh2

0.5

n=4 -10 -8 -6 -4 Log [sulfoximine 1 (M)]

Fig. 8. Sulfoximine 1-induced desensitization of IACh1 and IACh2. A.B. Like sulfoxaflor (see Fig. 7 for details), sulfoximine 1 (106 M) inhibited IACh2 completely but it inhibited IACh1 only partially, indicating that IACh1 consists of a low (IACh1L) and a high (IACh1H) sensitivity component. C. Concentrationeresponse relations for sulfoximine 1 inhibition of IACh1H and IACh2. From fits with the Hill equation (Eq. (1)) we estimated an IC50 for IACh2 of 3 109 M (1 109 Me7 109 M; nH ¼ 0.8 0.10) and 2 109 M (1 109 Me5 109 M, nH ¼ 1.0 0.11) for IACh1H.

Sulfoximine 2 (Fig. 9C): 5 109 M (2 109 Me15 109 M), nH ¼ 0.6 0.10, n ¼ 4. The concentrationeresponse relations for inhibition of IACh2 could be fit with a single binding site model with different IC50s for each sulfoximine: Sulfoxaflor (Fig. 7C): 2 109 M (1 109 Me5 109 M), nH ¼ 0.9 0.15, n ¼ 6. Sulfoximine 1 (Fig. 8C): 3 109 M (1 109 Me7 109 M), nH ¼ 0.8 0.10, n ¼ 4. Sulfoximine 2 (Fig. 9C): 2.3 109 M (0.9 109 Me8 109 M), nH ¼ 0.7 0.12, n ¼ 4. 4. Discussion The effects of imidacloprid, sulfoxaflor and two experimental sulfoximines on nicotinic receptors in stick insect neurons were studied. Bath application of the insecticides was used, to mimic the continued exposure experienced by the neurons during poisoning. When applied as pulses, these agonists can activate the receptors, but continuous exposure during poisoning is expected to result in desensitization and effective inhibition of cholinergic neurotransmission at much lower concentrations, because of the high affinity

IACh1H IACh1

0

IACh2

n=4

n=4

-8 -6 -10 -4 Log [sulfoximine 2 (M) ]

-6 -4 -10 -8 Log [sulfoximine 2 (M) ]

Fig. 9. Sulfoximine 2-induced desensitization of IACh1 and IACh2. A, B. Like sulfoxaflor (see Fig. 7 for details), sulfoximine 2 inhibited IACh2 completely, but it inhibited IACh1 only partially, indicating that IACh1 consists of a low (IACh1L) and a high (IACh1H) sensitivity component. C. Concentrationeresponse relations for sulfoximine 2 inhibition of IACh1H and IACh2. From fits with the Hill Equation (Eq. (1)) we estimated an IC50 for IACh2 of 3 109 M (0.9 109 Me8 109 M; nH ¼ 0.8 0.10) and 5 109 M (2 109 Me15 109 M, nH ¼ 0.6 0.10) for IACh1H.

of agonists for desensitized receptors (Salgado and Saar, 2004). Thus, while pulses of imidacloprid activated nACh1 receptors in stick insects with an EC50 of 5 105 M, and nACh2 receptors with an EC50 of 105 M (Oliveira et al., 2010), imidacloprid was found in the present study to inhibit both of these currents by more than 50% at 1012 M. In contrast to the American cockroach (Salgado and Saar, 2004), prolonged application of imidacloprid in the bath was never seen to activate currents in this study, suggesting that stick insects neurons do not possess nondesensitizing nAChRs. IACh1 and IACh2 are each composed of two components with differential sensitivity to imidacloprid. The high-sensitivity currents were both inhibited completely by 1012 M imidacloprid, while the lowsensitivity components were inhibited with IC50s of 81 and 2.6 nM, respectively. In contrast to imidacloprid, the three sulfoximines appeared to act equally well on both components of IACh2, so that the concentrationeresponse relation for desensitization of this current by all sulfoximines could be fit with a single binding site model. A component of IACh1 with the same time constant of


desensitization as IACh1L was insensitive to sulfoximines and was subtracted from total IACh1 to obtain the sensitive component, IACh1H, which was found to be inhibited by sulfoximines with IC50s between 2 and 7 nM. Thus, while all four of the desensitizing nACh currents found in the stick insect neurons were inhibited by imidacloprid at nM or lower concentrations, at least part of the desensitizing nACh current was completely insensitive to sulfoximines. Recently, studies were published that examined the displacement of imidacloprid binding from membranes of green peach aphids by sulfoximines (Watson et al., 2011; Zhu et al., 2010). In those studies, sulfoxaflor was relatively weak, displacing imidacloprid with an IC50 of 107 M. The same studies also reported that sulfoxaflor activated heterologously expressed nAChRs with high efficacy and it was postulated that despite its apparent low affinity for the receptors, the high efficacy was responsible for the high insecticidal activity (Watson et al., 2011; Zhu et al., 2010). This is clearly not the mode of action of sulfoximines in stick insects. Since stick insect nAChRs desensitize completely in the presence of sulfoximines and imidacloprid, the receptors would be functionally inhibited during poisoning, and efficacy would not be expected to play a role. Furthermore, in contrast to the heterologously expressed receptors studied by Watson et al. (2011) and Zhu et al. (2010), sulfoximines have very low efficacy on native nAChRs in stick insect neurons (Figs. 2 and 3). On the other hand, sulfoximines do indeed have high affinity for certain subtypes of nAChRs in stick insects, which could account for their activity. It seems possible that, like stick insects, aphids also have nAChRs with high affinity for sulfoximines, but that these were not measured under the conditions used by Zhu et al. (2010) and (Watson et al., 2011). In fact, at least two binding sites for imidacloprid have been observed in a number of species, including M. persicae (Lind et al., 1998), and the subunit composition of two imidacloprid-binding receptors in brown planthopper has recently been determined (Li et al., 2010). Imidacloprid and the three sulfoximines caused depressive rather than excitatory symptoms in stick insects. While nicotinic agonists are generally thought to produce excitatory symptoms in insects, this is not always the case. Tan et al. (2007) found that while nicotine, imidacloprid, thiacloprid and nitenpyram produced strong excitatory symptoms in American cockroaches, characterized by uncoordinated quivering, hyper-excitability and rapid spontaneous movements, wing flexing, violent body shaking and leg tremors, cockroaches exposed to clothianidin, dinotefuran or acetamiprid became immobile, with some postural changes (lowered head) and then became paralyzed and eventually died. While we did not study the link between symptoms and receptor effects in this study, it seems likely that the types of symptoms produced depends on receptor subtypes affected, and the functions of those subtypes in the particular species. In the present study, sulfoximines consistently caused ovipositor opening and dorsad bending of the abdomen, whereas imidacloprid caused these symptoms only in some insects. Furthermore, while imidacloprid and the sulfoximines decreased spontaneous movements, with imidacloprid this symptom was also associated with generalized weakness, which was not always the case with sulfoximines. These results highlight the importance of receptor subtypes in the mode of action of nicotinic agonist insecticides. Previous work in cockroaches indicated the presence of desensitizing and nondesensitizing nAChR subtypes (Salgado and Saar, 2004). The current work provides evidence for four desensitizing nAChR subtypes in stick insects, with no indication of the existence of nondesensitizing subtypes. Spinosad acts exclusively on nondesensitizing nAChRs (Salgado and Saar, 2004), and it is likely that neonicotinoids and other competitive nicotinic agonists such as

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sulfoximines act exclusively through desensitizing nAChRs, on which they gain several orders of magnitude in potency through the mechanism of desensitization (Salgado and Saar, 2004). Furthermore, the present work suggests that differential action on various desensitizing nAChR subtypes could be important in the action of various competitive nicotinic agonists, and this information might be important in resistance management, which is largely based on minimizing development of target site resistance through rotation of compounds acting at different target sites (IRAC, 2010). The observation that sulfoximines have differential actions on desensitizing IACh subtypes provides pharmacological tools that might contribute to future studies that attempt to better understand the synaptic and integrative properties of locomotory network component neurons. Acknowledgments We thank Helmut Wratil for excellent technical assistance. This work was supported by grants of the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) from the Brazilian Ministry of Education and the Deutscher Akademischer Austausch Dienst (DAAD) to EEO and grants of the Deutsche Forschungsgemeinschaft (DFG) to JS, AB and PK. References Burrows, M., 1996. The Neurobiology of an Insect Brain. Oxford University Press, Oxford. Büschges, A., Akay, T., Gabriel, J.P., Schmidt, J., 2008. Organizing network action for locomotion: insights from studying insect walking. Brain Res. Rev. 57, 162e171. Büschges, A., 1998. Inhibitory synaptic drive patterns motoneuronal activity in rhythmic preparations of isolated thoracic ganglia in the stick insect. Brain Res. 783, 262e271. Delpech, V.R., Ihara, M., Coddou, C., Matsuda, K., Sattelle, D.B., 2003. Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. Invert. Neurosci. 5, 29e35. EPA, 2008. Registration Eligibility Decision (RED) for Nicotine. United States Environmental Protection Agency. EPA, 2009. Nicotine: Product Cancellation Order, Federal Register. United States Environmental Protection Agency, 26695e26696 pp. Grauso, M., Reenan, R.A., Culetto, E., Sattelle, D.B., 2002. Novel putative nicotinic acetylcholine receptor subunit genes, Da5, Da6 and Da7 in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing. Genetics 160, 1519e1533. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv 391, 85e100. Ihara, M., Brown, L.A., Ishida, C., Okuda, H., Sattelle, D.B., Matsuda, K., 2006. Actions of imidacloprid, clothianidin and related neonicotinoids on nicotinic acetylcholine receptors of American cockroach neurons and their relationships with insecticidal potency. J. Pestic. Sci. 31, 35e40. IRAC, 2010. Insecticide Mode of Action Scheme. IRAC International MoA Working Group. http://www.irac-online.org/wp-content/uploads/2009/09/MoA-classification_v7.0. 4-5Oct10.pdf, 1e23 pp. Jeschke, P., Nauen, R., 2008. Neonicotinoids e from zero to hero in insecticide chemistry. Pest Manag. Sci. 64, 1084e1098. Langley, J.N., 1905. On the reaction of cells and of nerve endings to certain poisons chiefly as regards the reaction of striated muscle to nicotine and to curare. J. Physiol. 33, 374e413. Lansdell, S.J., Millar, N.S., 2002. Db3, an atypical nicotinic acetylcholine receptor subunit from Drosophila: molecular cloning, heterologous expression and coassembly. J. Neurochem. 80, 1009e1018. Lee, S.-J., Tomizawa, M., Casida, J.E., 2003. Nereistoxin and cartap neurotoxicity attributable to direct block of the insect nicotinic receptor/channel. J. Agric. Food Chem. 51, 2646e2652. Li, J., Shao, Y., Ding, Z., Bao, H., Liu, Z., Han, Z., Millar, N.S., 2010. Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid. Insect Biochem. Mol. Biol. 40, 17e22. Lind, R.J., Clough, M.S., Reynolds, S.E., Earley, F.G.P., 1998. [3H]Imidacloprid labels high- and low-affinity nicotinic acetylcholine receptor-like binding sites in the aphid Myzus persicae (Hemiptera: aphididae). Pestic. Biochem. Physiol. 62, 3e14. Loso, M.R., Nugent, B.M., Huang, J.X., Rogers, R.B., Zhu, Y., Renga, J.M., Hegde, V.B., Demark, J.J., 2007. Insecticidal N-substituted (6-haloalkylpyridin-3-yl)Alkyl Sulfoximines. PCT Pat. Appl. Publ.. WO 2007095229.

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