Calcium pathways such as cAMP modulate clothianidin action through activation of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors

NeuroToxicology 37 (2013) 127–133

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NeuroToxicology

Calcium pathways such as cAMP modulate clothianidin action through activation of a-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors Delphine Calas-List, Olivier List, Sophie Quinchard, Steeve H. Thany * Universite´ d’Angers, Laboratoire Re´cepteurs et Canaux Ioniques Membranaires (RCIM), UPRES EA 2647/USC INRA 1330, UFR de Sciences, 2 Bd. Lavoisier, 49045 Angers, France

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 February 2013 Accepted 22 April 2013 Available online 28 April 2013

Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of a-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors expressed in the cockroach dorsal unpaired median neurons. Clothianidin was able to act as an agonist of imidacloprid-insensitive nAChR2 receptor and internal regulation of cAMP concentration modulated nAChR2 sensitivity to clothianidin. In the present study, we demonstrated that cAMP modulated the agonist action of clothianidin via a-bungarotoxin-sensitive and insensitive receptors. Clothianidininduced current–voltage curves were dependent to clothianidin concentrations. At 10 mM clothianidin, increasing cAMP concentration induced a linear current–voltage curve. Clothianidin effects were blocked by 0.5 mM a-bungarotoxin suggesting that cAMP modulation occurred through abungarotoxin-sensitive receptors. At 1 mM clothianidin, cAMP effects were associated to abungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5 mM mecamylamine and 20 mM d-tubocurarine. In addition, we found that application of 1 mM clothianidin induced a strong increase of intracellular calcium concentration. These data reinforced the finding that calcium pathways including cAMP modulated clothianidin action on insect nicotinic acetylcholine receptors. We proposed that intracellular calcium pathways such as cAMP could be a target to modulate the mode of action of neonicotinoid insecticides. ß 2013 Elsevier Inc. All rights reserved.

Keywords: Insect Cockroach DUM neurons Nicotinic receptors Neonicotinoid Clothianidin Calcium cAMP

1. Introduction Insect nicotinic acetylcholine receptors (nAChRs) are pentameric receptors, formed by the combination of identical (homomeric) or different (heteromeric) subunits conferring distinct electrophysiological and pharmacological properties (Lansdell et al., 2012; Matsuda et al., 2001; Thany et al., 2007). They are involved in many processes such as rapid synaptic transmission, learning and memory (Gauthier, 2010; Gauthier et al., 2006) and are the main targets of neonicotinoid insecticides (Matsuda et al., 2005; Millar and Denholm, 2007; Thany et al., 2007; Tomizawa and Casida, 2003, 2009). Evidence of neonicotinoids action on insect nAChRs is based in part on electrophysiological studies (Ihara et al., 2007; Liu et al., 2009; Tan et al., 2007). Neonicotinoids such as clothianidin (CLO) are synthetic compounds that have been used as insecticides against agricultural and household pests (Uneme, 2011). They are reported to act as full or partial agonists on insect nAChRs affecting the central nervous system (Deglise et al., 2002; Tan et al., 2007).

* Corresponding author. Tel.: +33 241735213; fax: +33 241735215. E-mail address: [email protected] (S.H. Thany). 0161-813X/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2013.04.011

In our previous studies using cockroach Periplaneta americana, we have demonstrated that application of CLO onto the sixth abdominal ganglion resulted in a dose-dependent depolarization of cockroach cercal afferent/giant interneuron synapses which was not reversible, suggesting a strong desensitization of postsynaptic interneurons (Thany, 2009). CLO-induced currents were insensitive to muscarinic antagonist but were blocked by nicotinic antagonists such as a-bungarotoxin (a-Bgt), mecamylamine (Mec) and d-tubocurarine (d-TC). At extrasynaptic level, on dorsal unpaired median (DUM) neurons, CLO current amplitudes were reduced by a-Bgt and completely blocked by MLA, Mec and d-TC (Thany, 2009). Thus, we have suggested that CLO acted as a specific agonist of cockroach a-Bgt-sensitive and -insensitive nAChRs including nAChR1 and nAChR2. We also confirmed that DUM neurons expressed a-Bgt-insensitive nAChR subtypes which were blocked by Mec and d-TC (Calas-List et al., 2012). Two distinct subtypes of a-Bgt-insensitive nAChRs, named nAChR1 and nAChR2, were previously identified (Courjaret and Lapied, 2001). The inward currents mediated by these nAChRs differed from each other on the basis on their voltage dependence (nAChR1 being activated between 80 mV and 30 mV whereas nAChR2 was activated between 30 mV and +20 mV), selective pharmacological properties (nAChR1 was blocked by d-TC, and nAChR2

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was sensitive to Mec) and intracellular signaling pathways (Courjaret and Lapied, 2001). Moreover, nicotine-induced currents elicited by nAChR1 activation were modulated via a cAMP/PKA cascade and two distinct PKC isozymes (‘‘classical’’ and ‘‘novel’’ PKC) (Courjaret et al., 2003). We recently demonstrated that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential g (TRPg) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to neonicotinoid insecticides such as CLO (Bodereau-Dubois et al., 2012). We hypothesized that the calcium/CaM complex modulated the efficacy of neonicotinoid on nAChR2 via the regulation of adenylyl cyclase activation, which thereby regulated the level of intracellular 30 –50 -cyclic adenosine monophosphate (cAMP). This was confirmed by the finding that high concentration of cAMP increased approximately 10-fold the ED50 value of neonicotinoids (Bodereau-Dubois et al., 2012). Thus, activation of cAMP pathways can modulate the properties of insect nAChR sensitivity to neonicotinoids (Bodereau-Dubois et al., 2012). Nevertheless, according to our recent data, several questions remain: what is the effect of cAMP on neonicotinoid action at low and high concentrations? Is the biphasic current–voltage curve induced by CLO at low concentrations modulated by cAMP? For this purpose, we investigated the mechanisms leading to the ED50 increase via cAMP by testing the agonist effect of CLO on DUM neurons at different cAMP concentrations. 2. Materials and methods 2.1. Insects

micropipette (resistance 1.8 MV when filled with agonist) positioned in solution at a distance of 100 mm from the isolated cell body. In this study, control conditions were cAMP free solutions (in comparison with our previous data in (BodereauDubois et al., 2012)). 2.4. Intracellular calcium recordings For calcium imaging experiments, isolated DUM neurons were incubated in the dark with 10 mM Fura-2 pentakis(acetoxymethyl) ester (Fura-2 AM; Sigma Chemicals) for 60 min at 37 8C. After loading, cells were washed three times in saline. The glass coverslips were then mounted in a recording chamber connected to a gravity perfusion system. Drugs were applied as described above. Imaging experiments were performed with an inverted Nikon Eclipse Ti microscope (Nikon, Tokyo, JAPAN) equipped with a Lamdba DG4 wavelength switcher (Sutter instrument, Novato, CA, USA). Images were collected with an Orca-R2 CCD camera (Hamamatsu photonics, Shizuoka, Japan) and recorded in the computer with Imaging Workbench software (version 6, Indec BioSystems, Santa Clara, CA, USA). Experiments were carried out at room temperature. All drugs were purchased from Sigma (Sigma– Aldrich, France). 2.5. Statistical analysis For statistical analysis, one-way ANOVA and Bonferroni post hoc test were employed, using Prism program (GraphPAD Software, San Diego, CA). For each data, I/Imax were normalized according to the Imax recorded in the same experimental condition.

The experiments were carried out on DUM neuron cell bodies isolated from the dorsal midline of the terminal abdominal ganglion of the nerve cord from adult male cockroaches Periplaneta americana. 2.2. Cells isolation

2.3. Patch clamp recordings

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Signals were recorded using an Axopatch 200B amplifier (Axon instruments, Foster City, CA), connected to a computer with the pClamp software control (pClamp 10.0, Axon Instruments). The liquid junction potential between bath and internal pipette solution was compensated before the formation of a gigaOhm seal (>5 GV). Patch pipettes were pulled from borosilicate glass capillary tubes (GC 150T-10; Clark Electromedical Instruments, Harvard Apparatus) and had resistance ranging from 0.9 to 1 MV when filled with the standard pipette solution containing (in mM): NaCl 10; MgCl2 1; CaCl2 0.5; HEPES 10; ATPMg 1; EGTA 10; K aspartate 160; KF 10; and adjusted to pH 7.4 with KOH. CLO was applied by pneumatic pressure ejection (15 psig, 100 ms, Miniframe, Medical System Corporation, USA) through a glass

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Cells were isolated following enzymatic treatment and mechanical dissociation as previously described (Lapied et al., 1990). Cells were incubated overnight at 29 8C before electrophysiological experiments. CLO-induced inward currents were recorded using patch-clamp technique in the whole-cell recording configuration (Lapied et al., 1990). The Petri dish containing isolated cell bodies was placed onto the inverted microscope (CK2: Olympus), and continuously bathed with the standard extracellular solution (in mM: NaCl 200, KCl 3.1, MgCl2 4, CaCl2 5, sucrose 50, HEPES 10, pH 7.4 adjusted with NaOH) using a gravity perfusion system positioned at a distance of 100 mm from the cell body.

Fig. 1. Clothianidin (CLO) current–voltage curves at 10 mM (A) and 1 mM (B). Each square represents mean  SEM (N = 8).

D. Calas-List et al. / NeuroToxicology 37 (2013) 127–133

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-2.5 Fig. 2. Effect of cAMP concentrations on the current–voltage curves induced by 10 mM clothianidin. cAMP concentration was applied in patch pipette. (A) 10 mM, (B) 50 mM and (C) 100 mM cAMP. Each square represents mean  SEM (N = 8). For 0 cAMP, see Fig. 1A.

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Fig. 3. Effect of 10 mM clothianidin on a-bungarotoxin-sensitive and -insensitive nAChRs. (A) Effect of 10 mM clothianidin without pipette application of cAMP. (B) Typical examples of clothianidin-induced currents under 0.5 mM alphabungarotoxin (a-Bgt), 5 mM mecamylamine (Mec) and 20 mM d-tubocurarine (d-TC). Arrows indicate 100 ms pulse application of 10 mM clothianidin. Control (Ctl) represents clothianidin application without cAMP. (C) Histogram illustrating the decrease of clothianidin current amplitudes under 100 mM cAMP. Control (Ctl) corresponds to 100 mM cAMP. Data are mean  SEM. In each case N = 5, *p < 0.05 and **p < 0.001.

3. Results 3.1. Effect of cAMP variation on clothianidin-induced current amplitudes Pressure application of 10 mM CLO evoked a specific current– voltage curve which increased linearly between 90 mV and 30 mV and decreased between 30 mV and +20 mV (Thany, 2009). We have consequently suggested that these biphasic current–voltage curves could suggest a complex action of CLO on DUM neuron nAChRs (Thany, 2009). Interestingly, increasing CLO concentration from 10 mM to 1 mM resulted in a linear current– voltage curve (Fig. 1). These linear current–voltage curves were observed at 0.1 mM IMI (Courjaret and Lapied, 2001) and it was suggested that IMI acted as an agonist of a-Bgt-insensitive nAChR1 (Courjaret and Lapied, 2001). These data demonstrated that CLO induced current–voltage curves were dependent to its concentration. To study the involvement of cAMP concentration on CLOinduced currents, we have progressively increased intracellular

cAMP concentrations from 0 to 100 mM. We found that 10 mM CLO induced linear current–voltage curves under 50 mM and 100 mM cAMP (Fig. 2). These data suggested that at high concentrations, cAMP modulated the agonist action of CLO on DUM neurons. Following these data, we were interested in identifying cAMP effects through a-Bgt-sensitive and -insensitive nAChRs. Thus, we tested the effect of the nicotinic antagonists a-Bgt, Mec and d-TC because the biphasic current–voltage curve suggested the existence of a-Bgt-insensitive receptors, including both nAChR1 and nAChR2 (Courjaret et al., 2003; Courjaret and Lapied, 2001; Thany, 2009; Thany et al., 2008). As shown, at 50 mV holding potential (0.83  0.14 nA, N = 5), at low cAMP concentrations all currents were reduced by nicotinic antagonists. Data were 0.47  0.08 nA (p < 0.05, N = 5), 0.03  0.01 nA (p < 0.001, N = 5) and 0.10  0.03 nA (p < 0.001, N = 5) at 0 mM cAMP, respectively (Fig. 3A). These results were similar to our previous data and confirmed that at low CLO concentration, CLO effects occurred through activation of a-Bgtsensitive and -insensitive nAChRs (Thany, 2009). Interestingly, at 100 mM cAMP, CLO-induced current amplitudes were significantly

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-7.0 Fig. 4. Effect of cAMP concentration on the current–voltage curves induced by 1 mM clothianidin. cAMP concentration was applied in patch pipette. (A) 10 mM, (B) 50 mM and (C) 100 mM cAMP. Each square represents mean  SEM (N = 8). For 0 cAMP, see Fig. 1B.

reduced by 0.5 mM a-Bgt (0.14  0.0 nA, p < 0.05, N = 6, Fig. 3B and C). Similar results were observed, under co-application of 0.5 mM aBgt with 5 mM Mec (0.05  0.0 nA, p < 0.05, N = 6, Fig. 3B and C) and 20 mM d-TC (0.11  0.01 nA, p < 0.05, N = 6, Fig. 3B and C) suggesting an action through a-Bgt-sensitive receptors. 3.2. Effect of cAMP on high clothianidin concentration In a second set of experiments, we evaluated the effects of cAMP variation on high CLO concentration (Fig. 4). Significant results were found after pulse application of 1 mM CLO. At 50 mV holding potential (1.3  0.11 nA, N = 5), current amplitudes were 2.2  0.33 nA (at 10 mM cAMP, p < 0.05, N = 5, Fig. 5A), 2.7  0.25 nA (at 50 mM cAMP, p < 0.05, N = 5, Fig. 5A) and 2.3  0.5 nA (at 100 mM cAMP, p < 0.05, N = 5, Fig. 5A), respectively. These data demonstrated that at high CLO concentrations, cAMP variations induced an elevation of CLO current amplitudes which was

α-Bgt Fig. 5. Effect of 1 mM clothianidin under cAMP variation. (A) cAMP concentration was increased from 0 to 100 mM. Control (Ctl) represents 1 mM clothianidin. (B) Effect of 1 mM clothianidin under bath application of 0.5 mM a-Bgt. In both cases, a-Bgt did not reduce current induced by 1 mM clothianidin. All data are recorded at 50 mV holding potential. Data are mean  SEM. *p < 0.05. In each case, N = 8. Control (Ctl) represents 1 mM clothianidin application without cAMP.

not dose-dependent. We proposed that at high CLO concentrations, the influence of cAMP variation on CLO-induced current amplitudes was dependent on a-Bgt-insensitive receptors because under 0.5 mM a-Bgt, no significant difference was found between control (1.35  0.11 nA), and CLO current amplitudes under 0 mM cAMP (1.20  0.24 nA, N = 8, Fig. 5B) and 100 mM cAMP (1.15  0.16 nA, N = 8, Fig. 5B). In addition, we found that all currents were completely blocked by 5 mM Mec and 20 mM d-TC demonstrating the involvement of a-Bgt-insensitive receptors (Fig. 6). Thus, we confirmed that cAMP pathways differently modulated CLO action on DUM neuron nAChRs. 3.3. Effect of calcium on clothianidin-induced current amplitudes cAMP variation produced distinct effect on clothianidin currents indicating an implication of intracellular Ca2+ concentration. Thus, we have tested the agonist action of CLO following extracellular Ca2+ variation. DUM neurons were incubated with lower extracellular Ca2+ concentration (Thany et al., 2008). When the external Ca2+ was reduced to 0 mM Ca2+, we found a pronounced reduction of CLO-induced current amplitudes after pulse application of 10 mM CLO. Current amplitudes were reduced from 0.63  0.1 nA to 0.19  0.01 nA (p < 0.001, N = 6, Fig. 7A and B). Under 0.5 mM a-Bgt which blocked a-Bgt-sensitive nAChRs, currents were reduced to 0.28  0.02 nA (p < 0.001, N = 6, Fig. 7A

D. Calas-List et al. / NeuroToxicology 37 (2013) 127–133

and B). Similar results were found with the neonicotinoid insecticide imidacloprid (IMI). With 0 mM extracellular Ca2+ solution, data were 0.44  0.05 nA in control conditions and 0.17  0.03 nA at 10 mM IMI (p < 0.05, N = 6, Fig. 7C). In our conditions, we suggested that calcium influx through DUM neuron nAChRs modulated CLO current amplitudes. Indeed, we found that 1 mM CdCl reduced CLO-induced current amplitudes to 0.20  0.01 nA (p < 0.001, N = 6) and blocked all CLO currents with Ca2+ free extracellular solution (Fig. 7D). These data were in accordance with previous studies showing that decreasing extracellular Ca2+ concentration reduced nicotine-induced current amplitudes (Thany et al., 2008). Moreover, as illustrated in Fig. 8, we found that 10 mM and 1 mM pulse application of CLO differently resulted to an increase of intracellular Ca2+ concentration. At 1 mM CLO we found a strong increase of intracellular Ca2+ concentration compared to 10 mM CLO. We propose that pulse application of 10 mM or 1 mM CLO differently interact with cAMP through Ca2+ variation.

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4. Discussion 4.1. Complex effects of clothianidin on cockroach DUM neurons

Fig. 6. Current–voltage curves induced by 1 mM clothianidin. (A) Current amplitudes were blocked by 5 mM mecamylamine (Mec) and (B) 20 mM dtubocurarine (d-TC). Each data point represents mean  SEM (N = 5), **p < 0.001. In both cases Ctl, control represents 1 mM application of clothianidin.

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In this work, we have examined the effects of cAMP on CLOinduced currents, focussing on DUM neuron a-Bgt-sensitive and insensitive receptors. We found that neonicotinoid insecticide CLO induced distinct effects on DUM neuron nAChRs. The biphasic current–voltage curves induced by CLO were dependent to CLO concentrations. At high concentration, CLO induced a linear current–voltage curve which was associated to a-Bgt-sensitive and -insensitive receptors including nAChR1 and nAChR2. Indeed, it was previously suggested that the linear current–voltage curve

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Fig. 7. Effect of 10 mM neonicotinoids on DUM neuron nAChRs, under 0 Ca2+ concentration. (A) Ionic currents induced by 10 mM CLO under 0 Ca2+ and 0.5 mM aBgt. (B) Histograms illustrating the decrease of CLO-induced current amplitudes. Ctl represents 10 mM clothianidin. (C) Effects of 10 mM imidacloprid (Ctl, control represents 10 mM imidacloprid) under bath application of 0.5 mM a-Bgt and 0 Ca2+. (D) Effect of 10 mM clothianidin (Ctl) under bath application of 1 mM CdCl and 0 Ca2+. Data are mean  SEM. **p < 0.001, *p < 0.05. In each case, N = 8.

Fig. 8. Variation of intracellular calcium concentration after pulse application of 10 mM and 1 mM clothianidin. (A) Each trace represents the variation of intracellular calcium concentration after 100 ms pulse duration of 10 mM (lower trace) and 1 mM (upper trace) clothianidin. (B, C) Intracellular calcium variations, 2 s (B) and 50 s (C) (at the pic), after 10 mM clothianidin application (N = 10 cells). (D, E) Intracellular calcium variations, after 1 mM clothianidin application (N = 11 cells).

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Fig. 9. Possible mechanisms for the upregulation of DUM neuron nAChRs after clothianidin application. At high clothianidin concentration, there is a strong increase of intracellular calcium concentration, leading to an increase in the generation of cAMP from ATP by adenylyl cyclase. Ca2+ can also up-regulates the receptors leading to a high CLO current amplitude (Allosteric mechanisms). DUM neurons contain calcium channels that contribute to maintain Ca2+ level. At low clothianidin concentration, these pathways are poorly activated, leading to a low clothianidin effect. nAChR: nicotinic acetylcholine receptors; CaMKII: calmodulin dependent calcium kinase II; CaM: calmodulin; Ca2+: calcium; [Ca2+]i: intracellular calcium concentration; AC: adenylate cyclase; CCh: Ca2+ channels; ATP: adenosine triphosphate; a-Bgt-nAChRs: a-Bgt-sensitive receptors; nAChRs: a-Bgt-insensitive receptors.

induced by IMI was due to a-Bgt-insensitive nAChR1 (Courjaret and Lapied, 2001). We proposed that the balance between biphasic and linear current–voltage curves could be due to cAMP variations. Thus, cAMP could be an important regulatory mechanism offering an additional mechanism for modulation of insecticides action. 4.2. cAMP modulates clothianidin-induced currents We have shown that cAMP modulated the agonist action of CLO on cockroach DUM neuron nAChRs. At low CLO concentration, we suggested that cAMP modulation occurred through aBgt-sensitive receptors whereas at high concentration, this modulation was dependent to a-Bgt-insensitive receptors such as nAChR1 and nAChR2. Based on our results we hypothesized

that calcium entry into the cell following CLO application account in part for the modulation of CLO currents. Ca2+ entry could occur through a-Bgt-sensitive and insensitive receptors. Thus, at low CLO concentration, increasing cAMP concentration resulted in a linear current–voltage curve suggesting that nAChRs sensitivity to CLO is modulated by the activation of Ca2+ influx as previously demonstrated (Bodereau-Dubois et al., 2012). The increase of cAMP concentration is sufficient to induce a phosphorylation of nAChRs through PKA activity. At high CLO concentration, cAMP effects were associated to a strong increase of intracellular Ca2+ concentration, leading to an increase of CLO currents. This increase is completely blocked by nicotinic antagonist mecamylamine and d-tubocurarine suggesting that a-Bgt-insensitive receptors were involved in cAMP modulation of CLO-induced currents. Moreover, we proposed that cAMP influenced CLO action via a-Bgt-insensitive receptors, including nAChR1 and nAChR2 (Courjaret et al., 2003; Courjaret and Lapied, 2001; Thany et al., 2008). Indeed, we demonstrated that variations of cAMP concentration influence ion channels conformation (i.e. TRPg) and therefore modulated intracellular calcium concentration. These changes ultimately influenced nAChRs sensitivity to neonicotinoids (Bodereau-Dubois et al., 2012; Grolleau et al., 2006; Wicher et al., 2004). In the present study and taking into account our previous data, we demonstrated that the mode of action of CLO on insect nAChRs appeared more complex and is modulated by calcium pathways, including cAMP (Fig. 9). At DUM neuron membrane potential, intracellular calcium increase can activate calmodulin (CaM) or calcium/calmodulin dependent protein kinase II (CaMKII) which in turn induces a phosphorylation of nAChRs via PKA activity. At high CLO concentration, cAMP effect is associated to a strong increase of intracellular Ca2+ concentration, leading to an increase of CLO-induced currents. At low CLO concentration, there is no sufficient increase of intracellular Ca2+. It appears that a-Bgt-sensitive receptors are the major subtypes driving this effect. Thus, mechanisms leading to a basal change of intracellular Ca2+ concentration will change the effect of neonicotinoid insecticides. Conflict of interest None. References Bodereau-Dubois B, List O, Calas-List D, Marques O, Communal PY, Thany SH, et al. Transmembrane potential polarization, calcium influx, and receptor conformational state modulate the sensitivity of the imidacloprid-insensitive neuronal insect nicotinic acetylcholine receptor to neonicotinoid insecticides. J Pharmacol Exp Ther 2012;341:326–39. Calas-List D, List O, Thany SH. Nornicotine application on cockroach dorsal unpaired median neurons induces two distinct ionic currents: Implications of different nicotinic acetylcholine receptors. Neurosci Lett 2012;518:64–8. Courjaret R, Grolleau F, Lapied B. Two distinct calcium-sensitive and -insensitive PKC up- and down-regulate an alpha-bungarotoxin-resistant nAChR1 in insect neurosecretory cells (DUM neurons). Eur J Neurosci 2003;17:2023–34. Courjaret R, Lapied B. Complex intracellular messenger pathways regulate one type of neuronal alpha-bungarotoxin-resistant nicotinic acetylcholine receptors expressed in insect neurosecretory cells (dorsal unpaired median neurons). Mol Pharmacol 2001;60:80–91. Deglise P, Grunewald B, Gauthier M. The insecticide imidacloprid is a partial agonist of the nicotinic receptor of honeybee Kenyon cells. Neurosci Lett 2002;321:13–6. Gauthier M. State of the art on insect nicotinic acetylcholine receptor function in learning and memory. Adv Exp Med Biol 2010;683:97–115. Gauthier M, Dacher M, Thany SH, Niggebrugge C, Deglise P, Kljucevic P, et al. Involvement of alpha-bungarotoxin-sensitive nicotinic receptors in long-term memory formation in the honeybee (Apis mellifera). Neurobiol Learn Mem 2006;86:164–74. Grolleau F, Stankiewicz M, Kielbasiewicz E, Martin-Eauclaire MF, Lavialle C, De Vente J, et al. Indirect activation of neuronal noncapacitative Ca2+ entry is the final step involved in the neurotoxic effect of Tityus serrulatus scorpion beta-toxin. Eur J Neurosci 2006;23:1465–78.

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