Isolation and electrophysiological recording of Ixodes ricinus synganglion neurons.

The central nervous system of hard ticks (Ixodidae) consists of a concentrated merged nerve mass known as the synganglion. Although knowledge of tick neurobiology has dramatically improved over the last two decades, this is the first time that isolation and electrophysiological recordings have been carried out on tick neurons from the synganglion. Method: We developed a simple protocol for synganglion neuron isolation and used a whole-cell patch clamp to measure ionic currents induced by acetylcholine, nicotine and muscarine. Relatively large neurons (∼ 25 µm and âˆ¼ 35 µm) were isolated and 1 mM acetylcholine was used to induce strong inward currents of -0.38 ± 0.1 nA and - 1.04 ± 0.1 nA, respectively, with the corresponding cell capacitances being at around 142 pF and 188 pF. In addition, successive application of 1 mM acetylcholine through ∼25 µm and âˆ¼ 35 µm cells for increasing amounts of time resulted in a rapid reduction in current amplitudes. We also found that acetylcholine-evoked currents were associated with a reversible increase in intracellular calcium levels for each neuronal type. In contrast, 1 mM muscarine and nicotine induced a strong and non-reversible increase in intracellular calcium levels. This study serves as a proof of concept for the mechanical isolation of tick synganglion neurons followed by their electrophysiological recording. This approach will aid investigations into the pharmacological properties of tick neurons and provides the tools needed for the identification of drug-targeted sites and effective tick control measures.

Identification of sulfonamide compounds active on the insect nervous system: Molecular modeling, synthesis and biological evaluation.

Insect nicotinic acetylcholine receptors (nAChRs) are a recognized target for insecticide design. In this work, we have identified, from a structure-based approach using molecular modeling tools, ligands with potential selective activity for pests versus pollinators. A high-throughput virtual screening with the Openeye software was performed using a library from the ZINC database, thiacloprid being used as the target structure. The top sixteen molecules were then docked in α6 cockroach and honeybee homomeric nAChRs to check from a theoretical point of view relevant descriptors in favor of pest selectivity. Among the selected molecules, one original sulfonamide compound has afterward been synthesized, together with various analogs. Two compounds of this family have been shown to behave as activators of the cockroach cholinergic synaptic transmission.

Effect of the combination of DEET and flupyradifurone on the tick Ixodes ricinus: Repellency bioassay and pharmacological characterization using microtransplantation of synganglion membranes.

Ticks are vectors of many human and animal pathogens, and represent a major threat to public health. In recent years, an increase in tick-borne diseases has been observed, and new strategies are therefore needed in order to control tick numbers and reduce human tick bites. In the present study, we adapted the previous tick repellency bioassay based on the exploration behavior of the tick, using the ToxTrac software and video-tracking, to compare the repellent effect of two compounds on the tick Ixodes ricinus: N,N-diethyl-methyl-m-toluamide (DEET), and butenolide, flupyradifurone (FLU). We found that when applied alone, 10% DEET or FLU have no/or low repellency effect. But, the combination of both 10% DEET and FLU demonstrated a significant repellency effect against I. ricinus, similar to the repellency of 20% DEET. Using membrane microtransplantation, we evaluated the effect of DEET and FLU on native acetylcholine receptors expressed on the tick synganglion. We found that DEET has no effect on acetylcholine-evoked currents, but significantly reduced nicotine-induced current amplitudes. FLU induced an ionic current but was not able to reduce acetylcholine or nicotine evoked currents. The combination of both DEET and FLU strongly reduced nicotine-evoked currents. Finally, we demonstrated that our recording device for repellency, as well as the use of membrane microtransplantation, could be used as methods to study the mode of action of active compounds on ticks.

Calmidazolium induces a decrease in nicotine-induced currents and intracellular calcium levels after pulse application of nicotine onto insect neurosecretory cells.

Dorsal unpaired median (DUM) neurons, are a class of insect neurosecretory cells, which are involved in the control of several functions, such as excretion and reproduction, or the release of neurohormones. Previous studies demonstrated that they express different nicotinic acetylcholine receptor subtypes, in particular α-bungarotoxin-insensitive receptors, with nAChR1 and nAChR2 subtypes. Here, we demonstrated that pulse application of 1 mM nicotine (300 ms pulse duration) induced inward currents which were reduced under bath application of 15 µM calmidazolium, a calmodulin inhibitor. Bath application of 0.5 µM α-bungarotoxin had no effect on calmidazolium action, suggesting that it could have an indirect effect through α-bungarotoxin-insensitive receptors. Indeed, nicotine-evoked currents were reduced by 10 µM d-tubocurarine, and completely blocked by 5 µM mecamylamine, which affected nAChR1 and nAChR2 subtypes, respectively. Our results demonstrated that nAChR2 subtypes are involved in the indirect effect of calmidazolium. Moreover, we found that this calmidazolium effect was associated to a strong reduction in intracellular calcium levels after pulse application of 1 mM nicotine. Thus, compared to previous studies on mammalian cells, calmidazolium did not cause an increase in intracellular calcium levels in DUM neurons, suggesting that different calcium mechanisms are involved in the calmidazolium effect.

Mode of Action of Neonicotinoid Insecticides Imidacloprid and Thiacloprid to the Cockroach Pameα7 Nicotinic Acetylcholine Receptor.

The functional expression of the cockroach Pameα7 nicotinic acetylcholine receptor subunit has been previously studied, and was found to be able to form a homomeric receptor when expressed in Xenopus laevis oocytes. In this study, we found that the neonicotinoid insecticide imidacloprid is unable to activate the cockroach Pameα7 receptor, although thiacloprid induces low inward currents, suggesting that it is a partial agonist. In addition, the co-application or 5 min pretreatment with 10 µM imidacloprid increased nicotine current amplitudes, while the co-application or 5 min pretreatment with 10 µM thiacloprid decreased nicotine-evoked current amplitudes by 54% and 28%, respectively. This suggesting that these two representatives of neonicotinoid insecticides bind differently to the cockroach Pameα7 receptor. Interestingly, the docking models demonstrate that the orientation and interactions of the two insecticides in the cockroach Pameα7 nAChR binding pocket are very similar. Electrophysiological results have provided evidence to suggest that imidacloprid and thiacloprid could act as modulators of the cockroach Pameα7 receptors.

Nicotinic acetylcholine receptors in the synganglion of the tick Ixodes ricinus: Functional characterization using membrane microtransplantation.

Nicotinic acetylcholine receptors are an important class of excitatory receptors in the central nervous system of arthropods. In the ticks Ixodes ricinus, the functional and pharmacological properties of nicotinic receptors located in their neurons are still unknown. The objective of this study was to characterize the pharmacological properties of tick nicotinic receptors using membrane microtransplantation in Xenopus laevis oocytes and two-electrodes voltage clamp method. The membranes microtransplanted were extracted from the tick synganglion. We found that oocytes microtransplanted with tick synganglion membranes expressed nicotinic acetylcholine receptor subtypes which were activated by acetylcholine (1 mM) and nicotine (1 mM). Currents induced by pressure application of acetylcholine and nicotine were diminished by 10 nM α-bungarotoxin and methyllycaconitine, suggesting that they expressed two subtypes of nicotinic receptors, α-bungarotoxin-sensitive and -insensitive, respectively. In addition, we found that nicotine receptors expressed in the synganglion membranes were poorly sensitive to the neonicotinoid insecticides clothianidin (CLT), imidacloprid (IMI), acetamiprid (ACE) and thiamethoxam (TMX), in agreement with their lack of activity as acaricides. Interestingly, current amplitudes were strongly potentialized in the presence of 1 µM PNU-120596. CLT was more active as an agonist than IMI, TMX and ACE. Finally, we demonstrated that microtransplantation of purified membrane from the tick synganglion can be a valuable tool for the development and screening of compounds targeting tick nicotinic acetylcholine receptor subtypes.

Synergic effect of a quinuclidine benzamide complexed with borane, the LMA10233, in combination with seven pesticides.

Some quinuclidine benzamide compounds have been found to modulate nicotinic acetylcholine receptors in both mammals and insects. In particular, the quaternarization of 3-amino quinuclidine benzamide derivatives with dichloromethane gave charged N-chloromethylated quinuclidine compounds, disclosing an antagonist profile on homomeric α7 nAChRs. Here, we synthesized and studied the toxicological effect of LMA10233, a quinuclidine-borane complex analogue, the LMA10233, on the pea aphid Acyrthosiphon pisum and found that LMA10233 only exhibit proper toxicity on A. pisum larvae when applied in concentrations of over 10 µg/ml. We assessed the ability of LMA10233 to enhance the toxicity of different insecticides. When a sublethal concentration of LMA10233 was combined with the LC10 of each compound, we found a strong increase in toxicity at 24 h and 48 h of exposure for clothianidin, fipronil and chlorpyrifos, and only at 24 h for imidacloprid, acetamiprid and deltamethrin. However, when the pesticide was used at the LC50, only acetamiprid showed a synergistic effect with LMA10233. When the concentration of LMA10233 was decreased, we found that up to 80-90% of mortality was obtained due to the synergism between acetamiprid and LMA10233. No similar effect was observed with other insecticides. We conclude that such quinuclidine-borane complex compounds could increase the toxic effect of insecticides at low concentrations.

Effects of the DAG analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents through α-bungarotoxin-insensitive nicotinic acetylcholine receptors expressed on cockroach neurosecretory cells.

We previously demonstrated that the cockroach α-bungarotoxin-sensitive nicotinic acetylcholine receptors, nAChR1 and nAChR2 subtypes, are differently sensitive to intracellular calcium pathways. Here, using whole cell patch-clamp recordings, we studied the effects of the diacylglycerol (DAG) analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents under an α-bungarotoxin treatment. Our results demonstrated that DiC8 reduced nicotine and clothianidin evoked currents. 10 µM DiC8 suppressed the increase in nicotine-induced currents which was brought about by application of 5 mM caffeine or 9 mM Ca2+, whereas DiC8 did not affect the decrease in nicotine-induced currents induced by BAPTA. Similarly, bath application of caffeine or 9 mM Ca2+ did not change the clothianidin effects, and the amplitude of clothianidin-induced currents was not affected. However, co-application of both 10 µM DiC8 with 9 mM Ca2+, caffeine or BAPTA reduced clothianidin current amplitudes. We conclude that nicotine and clothianidin differently modulate nAChR1 and nAChR2 subtypes under DiC8 treatment, and that nicotine activates nAChR1, whereas clothianidin activates both nAChR1 and nAChR2 subtypes.

Mode of action of sulfoxaflor on α-bungarotoxin-insensitive nAChR1 and nAChR2 subtypes: Inhibitory effect of imidacloprid.

Cockroach neurosecretory cells, dorsal unpaired median (DUM) neurons, express two distinct α-bungarotoxin-insensitive nicotinic acetylcholine receptor subtypes, nAChR1 and nAChR2 which are differently sensitive to the neonicotinoid insecticides and intracellular calcium pathways. The aim of this study is to determine whether sulfoxaflor acts as an agonist of nAChR1 and nAChR2 subtypes. We demonstrated that 1 mM sulfoxaflor induced high current amplitudes, compared to acetylcholine, suggesting that it was a full agonist of DUM neuron nAChR subtypes. Sulfoxaflor evoked currents were not inhibited by the nicotinic acetylcholine receptor antagonist d-tubocurarine (dTC) which reduced nAChR1. But, sulfoxaflor evoked currents were reduced in the presence of 5 µM mecamylamine which is known to reduce nAChR2 subtype. Interestingly, when 1 µM imidacloprid was added in the extracellular solution, sulfoxaflor-induced currents were significantly suppressed. Moreover, when extracellular calcium concentration was increased, bath application of 1 µM imidacloprid partially reduced sulfoxaflor activated currents when nAChR1 was inhibited with 20 µM dTC and completely suppressed sulfoxaflor currents when nAChR2 was inhibited with 5 µM mecamylamine. Our data demonstrated therefore that sulfoxaflor activates both nAChR1 and nAChR2 subtypes.

Permethrin enhances the agonist activity of dinotefuran on insect cholinergic synaptic transmission and isolated neurons.

Insect resistance mechanisms against pesticides lead to the development and the search of new pesticide combinations in order to delay the resistance. The combination of neonicotinoids with pyrethroids was currently proposed but the mode of action of these compounds at synaptic and extrasynaptic levels needs to be further explored. In the present study, we evaluated the effect of the combination of two insecticides, permethrin and dinotefuran, on cockroach cholinergic synaptic transmission and on isolated cell bodies. We first found that combination of 5 µM permethrin and dinotefuran enhances depolarization of the sixth abdominal ganglion compared to dinotefuran alone, without an inhibition of the spontaneous activity. However, a pretreatment with 1 µM dinotefuran or permethrin before bath application of the mixture inhibits the ganglionic depolarization. Compared to permethrin, 1 µM dinotefuran induces a persistent enhancement of spontaneous activity. Interestingly, at extrasynaptic level, using dorsal unpaired median neurons and Kenyon cells, we found that combination of both 1 µM dinotefuran and permethrin resulted in an increase of the mixture-induced current amplitudes. Pretreatment with 1 µM dinotefuran strongly decreases the currents whereas permethrin induces a time-dependent inhibition. These data demonstrate that the combination of dinotefuran and permethrin enhances the effect of dinotefuran.

The Differential Effect of Low-Dose Mixtures of Four Pesticides on the Pea Aphid Acyrthosiphon pisum.

The modes of action of most insecticides are known, but little information exists regarding the toxicological interactions involving insecticide mixtures at low doses. The effects of mixtures of four insecticides were investigated using LC10 values (concentration leading to 10% mortality), acetamiprid (ACE, 0.235 µg/mL), chlorpyriphos (CHL, 107.0 µg/mL), deltamethrin (DEL, 5.831 µg/mL), and fipronil (FIP, 3.775 µg/mL) on the larvae of the pea aphid, Acyrthosiphon pisum. After 24 h exposure, 6 of the 11 tested combinations, DEL/FIP, ACE/DEL, CHL/FIP, ACE/DEL/FIP, ACE/CHL/FIP, and ACE/DEL/CHL/FIP, were toxic through an additive effect. Four combinations, ACE/FIP, DEL/CHL, ACE/CHL, and ACE/DEL/CHL had a synergistic effect, whereas only one DEL/CHL/FIP showed an antagonistic effect. The toxic effect of these mixtures was confirmed after 48 h of exposure, revealing an enhanced toxicity of CHL, DEL, and FIP in combination with ACE. We suggest that an insect pest management strategy should be evaluated in the future using different combinations of insecticides.

cGMP/cGMP-dependent protein kinase pathway modulates nicotine-induced currents through the activation of α-bungarotoxin-insensitive nicotinic acetylcholine receptors from insect neurosecretory cells.

Insect neurosecretory cells, called dorsal unpaired median neurons, are known to express two α-bungarotoxin-insensitive nicotinic acetylcholine receptor (nAChR) subtypes, nAChR1 and nAChR2. It was demonstrated that nAChR1 was sensitive to cAMP/cAMP-dependent protein kinase (PKA) regulation, resulting in a modulation of nicotine currents. In this study, we show that cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway modulates nicotine-induced currents, as increased cGMP affects the second compound of the biphasic current-voltage curve, corresponding to the nAChR2 receptors. Indeed, maintaining the guanosine triphosphate level with 100 µM guanosine triphosphate-γ-S increased nicotine currents through nAChR2. We also demonstrated that inhibition of PKG activity with 0.2 µM (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT5823), a PKG specific inhibitor, reduced nicotine-induced current amplitudes. KT5823 effect on nicotine currents is associated with calcium (Ca(2+) ) activity because inhibition of Ca(2+) concentration with cadmium chloride (CdCl2 ) abolished KT5823-induced inhibition mediated by nAChR2. However, specific inhibition of nitric oxide-guanylyl cyclase (GC) complex by 10 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) significantly increased nicotine-induced current amplitudes on both nAChR1 and nAChR2. These results suggest that nicotine-induced currents mediated by both α-bungarotoxin-insensitive nAChR1 and nAChR2 are coupled to the cGMP/PKG pathway. We propose that nicotinic acetylcholine receptor activation induces an increase in intracellular calcium (Ca(2+) ) concentration. Elevation of intracellular Ca(2+) results in the formation of Ca(2+) -calmodulin (CaM) complex, which activates guanylyl cyclase (GC) and/or adenylyl cyclase (AC). Ca(2+) -CaM complex could activate Ca(2+) calmodulin kinase II which could directly or indirectly modulate the nicotinic response. The mechanisms by which cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) interact remain unclear. We demonstrate that nicotine-induced currents are coupled to the cGMP/PKG pathway.

Molecular recognition of thiaclopride by Aplysia californica AChBP: new insights from a computational investigation.

The binding of thiaclopride (THI), a neonicotinoid insecticide, with Aplysia californica acetylcholine binding protein (Ac-AChBP), the surrogate of the extracellular domain of insects nicotinic acetylcholine receptors, has been studied with a QM/QM' hybrid methodology using the ONIOM approach (M06-2X/6-311G(d):PM6). The contributions of Ac-AChBP key residues for THI binding are accurately quantified from a structural and energetic point of view. The importance of water mediated hydrogen-bond (H-bond) interactions involving two water molecules and Tyr55 and Ser189 residues in the vicinity of the THI nitrile group, is specially highlighted. A larger stabilization energy is obtained with the THI-Ac-AChBP complex compared to imidacloprid (IMI), the forerunner of neonicotinoid insecticides. Pairwise interaction energy calculations rationalize this result with, in particular, a significantly more important contribution of the pivotal aromatic residues Trp147 and Tyr188 with THI through CH···π/CH···O and π-π stacking interactions, respectively. These trends are confirmed through a complementary non-covalent interaction (NCI) analysis of selected THI-Ac-AChBP amino acid pairs.

Pharmacological profile of zacopride and new quaternarized fluorobenzamide analogues on mammalian α7 nicotinic acetylcholine receptor.

From quaternarization of quinuclidine enantiomers of 2-fluoro benzamide LMA10203 in dichloromethane, the corresponding N-chloromethyl derivatives LMA10227 and LMA10228 were obtained. Here, we compared the agonist action of known zacopride and its 2-fluoro benzamide analogues, LMA10203, LMA10227 and LMA10228 against mammalian homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We found that LMA10203 was a partial agonist of α7 receptor with a pEC50 value of 4.25 ± 0.06 µM whereas LMA10227 and LMA10228 were poorly active on α7 homomeric nicotinic receptor. LMA10227 and LMA10228 were identified as antagonists of acetylcholine-induced currents with IC50 values of 28.4 µM and 39.3 µM whereas LMA10203 and zacopride possessed IC50 values of 8.07 µM and 7.04 µM, respectively. Moreover, despite their IC50 values, LMA10227 was the most potent inhibitor of nicotine-induced current amplitudes (65.7 ± 2.1% inhibition). LMA10203 and LMA10228 had the same inhibitory effects (26.5 ± 7.5% and 33.2 ± 4.1%, respectively), whereas zacopride had no significant inhibitory effect (4.37 ± 4%) on nicotine-induced responses. Our results revealed different pharmacological properties between the four compounds on acetylcholine and nicotine currents. The mode of action of benzamide compounds may need to be reinterpreted with respect to the potential role of α7 receptor.

Molecular features and toxicological properties of four common pesticides, acetamiprid, deltamethrin, chlorpyriphos and fipronil.

Structural features and selected physicochemical properties of four common pesticides: acetamiprid (neonicotinoid), chlorpyriphos (organophosphate insecticide), deltamethrin (pyrethroid) and fipronil (phenylpyrazole) have been investigated by Density Functional Theory quantum chemical calculations. The high flexible character of these insecticides is revealed by the numerous conformers obtained, located within a 20kJmol(-1) range in the gas phase. In line with this trend, a redistribution of the energetic minima is observed in water medium. Molecular electrostatic potential calculations provide a ranking of the potential interaction sites of the four insecticides. The theoretical studies reported in the present work are completed by comparative toxicological assays against three aphid strains. Thus, the same toxicity order for the two susceptible strains Myzus persicae 4106A and Acyrthosiphon pisum LSR1: acetamiprid>fipronil>deltamethrin>chlorpyriphos is revealed. In the resistant strain M. persicae 1300145, the toxicity order is modified: acetamiprid>fipronil>chlorpyriphos>deltamethrin. Interestingly, the strain 1300145 which is known to be resistant to neonicotinoids, is also less sensitive to deltamethrin, chlorpyriphos and fipronil.

Imidacloprid and thiacloprid neonicotinoids bind more favourably to cockroach than to honeybee α6 nicotinic acetylcholine receptor: insights from computational studies.

The binding interactions of two neonicotinoids, imidacloprid (IMI) and thiacloprid (THI) with the extracellular domains of cockroach and honeybee α6 nicotinic acetylcholine receptor (nAChR) subunits in an homomeric receptor have been studied through docking and molecular dynamics (MD) simulations. The binding mode predicted for the two neonicotinoids is validated through the good agreement observed between the theoretical results with the crystal structures of the corresponding complexes with Ac-AChBP, the recognized structural surrogate for insects nAChR extracellular ligand binding domain. The binding site of the two insect α6 receptors differs by only one residue of loop D, a serine residue (Ser83) in cockroach being replaced by a lysine residue (Lys108) in honeybee. The docking results show very close interactions for the two neonicotinoids with both α6 nAChR models, in correspondence to the trends observed in the experimental neonicotinoid-Ac-AChBP complexes. However, the docking parameters (scores and energies) are not significantly different between the two insect α6 nAChRs to draw clear conclusions. The MD results bring distinct trends. The analysis of the average interaction energies in the two insects α6 nAChRs shows indeed better affinity of neonicotinoids bound to α6 cockroach compared to honeybee nAChR. This preference is explained by tighter contacts with aromatic residues (Trp and Tyr) of the binding pocket. Interestingly, the non-conserved residue Lys108 of loop D of α6 honeybee nAChR interacts through van der Waals contacts with neonicotinoids, which appear more favourable than the direct or water mediated hydrogen-bond interaction between the OH group of Ser83 of α6 cockroach nAChR and the electronegative terminal group of the two neonicotinoids (nitro in IMI and cyano in THI). Finally, in both insects nAChRs, THI is consistently found to bind more favourably than IMI.

Inhibition of PaCaMKII-E isoform in the dorsal unpaired median neurosecretory cells of cockroach reduces nicotine- and clothianidin-induced currents.

Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII), which transduces the signal into downstream effects. We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms, and only PaCaMKII-E isoform is specifically expressed in the dorsal unpaired median neurosecretory cells. In the present study, using antisense oligonucleotides, we demonstrated that PaCaMKII-E isoform inhibition reduced nicotine-induced currents through α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptor subtypes. Specifically, PaCaMKII-E isoform is sufficient to repress nicotinic current amplitudes as a result of its depression by antisense oligonucleotides. Similar results were found using the neonicotinoid insecticide clothianidin, which acted as a full agonist of dorsal unpaired median neuron nicotinic acetylcholine receptors. Clothianidin current amplitudes are strongly reduced under bath application of PaCaMKII-E antisense oligonucleotides but no significant results are found with α-bungarotoxin co-applied, demonstrating that CaMKII-E isoform affects nicotine currents through α-bungarotoxin-sensitive and -insensitive receptor subtypes whereas clothianidin currents are reduced via α-bungarotoxin-insensitive receptors. In addition, we found that intracellular calcium increase induced by nicotine and clothianidin were reduced by PaCaMKII-E antisense oligonucleotides, demonstrating that intracellular calcium increase induced by nicotine and clothianidin are affected by PaCaMKII-E inhibition. Cellular responses to Ca(2+) require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII). We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms and only PaCaMKII-E isoform was specifically expressed in the dorsal unpaired median neurosecretory cells. Here we show that specific inhibition of PaCaMKII-E isoform is associated with a decrease in nicotine- and clothianidin-induced currents. In addition, analysis of calcium changes demonstrates that PaCaMKII-E inhibition induces a decrease in intracellular calcium concentration.

Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations.

We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors.

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

Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of α-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 α-bungarotoxin-sensitive and insensitive receptors. Clothianidin-induced current-voltage curves were dependent to clothianidin concentrations. At 10 µM clothianidin, increasing cAMP concentration induced a linear current-voltage curve. Clothianidin effects were blocked by 0.5 µM α-bungarotoxin suggesting that cAMP modulation occurred through α-bungarotoxin-sensitive receptors. At 1 mM clothianidin, cAMP effects were associated to α-bungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5 µM mecamylamine and 20 µM d-tubocurarine. In addition, we found that application of 1mM 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.

[Neurotoxicity of pesticides: its relationship with neurodegenerative diseases]. / Neurotoxicité des pesticides - Quel impact sur les maladies neurodégénératives ?

Several epidemiological studies suggest that pesticides could lead to neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Among pesticides, insecticides appear more neurotoxic than others but the neurotoxic mechanisms leading to adverse health effects remain unclear. The currently used pesticides such as rotenone and paraquat could disrupt mitochondrial bioenergetic function, reactive oxygen metabolism, redox function and promote α-synuclein aggregation. In addition, recent studies demonstrate that genetic susceptibility to Parkinson's disease could monitor pesticide susceptibility, as demonstrated for polymorphisms in pesticide metabolizing enzymes that are involved in organophosphorus sensitivity.