Bruno Lapied

Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet

BackgroundN,N-Diethyl-3-methylbenzamide (deet) remains the gold standard for insect repellents. About 200 million people use it every year and over 8 billion doses have been applied over the past 50 years. Despite the widespread and increased interest in the use of deet in public health programmes, controversies remain concerning both the identification of its target sites at the olfactory system and its mechanism of toxicity in insects, mammals and humans. Here, we investigated the molecular target site for deet and the consequences of its interactions with carbamate insecticides on the cholinergic system.ResultsBy using toxicological, biochemical and electrophysiological techniques, we show that deet is not simply a behaviour-modifying chemical but that it also inhibits cholinesterase activity, in both insect and mammalian neuronal preparations. Deet is commonly used in combination with insecticides and we show that deet has the capacity to strengthen the toxicity of carbamates, a class of insecticides known to block acetylcholinesterase.ConclusionThese findings question the safety of deet, particularly in combination with other chemicals, and they highlight the importance of a multidisciplinary approach to the development of safer insect repellents for use in public health.

Indoxacarb, an oxadiazine insecticide, blocks insect neuronal sodium channels

Decarbomethoxyllated JW062 (DCJW), the active component of a new oxadiazine insecticide DPX‐JW062 (Indoxacarb), was tested on action potentials and the inward sodium current recorded from short‐term cultured dorsal unpaired median neurones of the cockroach Periplaneta americana. Under whole‐cell current‐clamp conditions, 100 nM DCJW reduced the amplitude of action potentials and induced a large hyperpolarization of the resting membrane potential associated with a 41% increase in input resistance. In voltage‐clamp, DCJW resulted in a dose‐dependent inhibition (IC50 28 nM) of the peak sodium current. Based on IC50 values, the effect of DCJW was about 10 fold less potent than tetrodotoxin (TTX) but 1000 fold more potent than the local anaesthetic lidocaine. DCJW (100 nM) was without effect on activation properties of the sodium current, reversal potential, voltage dependence of sodium conductance and on both fast and slow steady‐state inactivations. TTX (2 nM) resulted in 48% inhibition of the peak inward sodium current. Co‐application of TTX (2 nM) with various concentrations of DCJW produced an additional inhibition of the peak inward current, indicating that the blocking actions of DCJW and TTX were distinct. Co‐application of lidocaine (IC50 30 μM) with various concentrations of DCJW produced a reduction of the apparent potency of DCJW, suggesting that DCJW and lidocaine acted at the same site. DCJW (100 nM) did not affect inward calcium or outward potassium currents. This study describes, for the first time, the action on insect neuronal voltage‐dependent sodium channels of Indoxacarb, a new class of insecticides.

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels ☆

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.

Synergy between repellents and non-pyrethroid insecticides strongly extends the efficacy of treated nets against Anopheles gambiae

BackgroundTo manage the kdr pyrethroid-resistance in Anopheline malaria vectors, new compounds or new strategies are urgently needed. Recently, mixing repellents (DEET) and a non-pyrethroid insecticide (propoxur) was shown to be as effective as deltamethrin, a standard pyrethroid, under laboratory conditions, because of a strong synergy between the two compounds. In the present study, the interactions between two repellents (DEET and KBR 3023) and a non-pyrethroid insecticide (pyrimiphos methyl or PM) on netting were investigated. The residual efficacy and the inhibition of blood feeding conferred by these mixtures were assessed against Anopheles gambiae mosquitoes.MethodsDEET and KBR 3023 were mixed with pyrimiphos methyl (PM), a organophosphate (OP) insecticide. The performance of mono- and bi-impregnated nets against adult mosquitoes was assessed using a miniaturized, experimental hut system (laboratory tunnel tests) that allows expression of behavioural responses to insecticide, particularly the mortality and blood feeding effects.ResultsBoth mixtures (PM+DEET and PM+KBR3023) induced 95% mortality for more than two months compared with less than one week for each compound used alone, then reflecting a strong synergy between the repellents and PM. A similar trend was observed with the blood feeding rates, which were significantly lower for the mixtures than for each component alone.ConclusionSynergistic interactions between organophosphates and repellents may be of great interest for vector control as they may contribute to increase the residual life of impregnated materials and improve the control of pyrethroid-resistance mosquitoes. These results prompt the need to evaluate the efficacy of repellent/non-pyrethroid insecticide mixtures against field populations of An. gambiae showing high level of resistance to Ops and pyrethroids.

Two distinct calcium-sensitive and -insensitive PKC up- and down-regulate an α-bungarotoxin-resistant nAChR1 in insect neurosecretory cells (DUM neurons)

While there is mounting knowledge about the structure and diversity of insect neuronal nicotinic acetylcholine receptors, less attention has been directed towards their intracellular regulation by calcium‐mediated activation or inhibition of protein phosphorylation. The main goal of this work was to delineate the chain of molecular events that lead to the up‐ and down‐regulation by two protein kinase Cs of an insect neuronal α‐bungarotoxin‐resistant nicotinic acetylcholine receptor (called nAChR1). The native nicotinic acetylcholine receptor intracellular regulation was studied on dissociated adult dorsal unpaired median neurons isolated from the terminal abdominal ganglion of the cockroach Periplaneta americana using whole‐cell patch‐clamp technique and calcium imaging. We report that under 0.5 µmα‐bungarotoxin treatment, the inward current produced by pressure ejection application of nicotine onto the cell body was differentially sensitive to specific protein kinase C activators and inhibitors. The phorbol ester PMA produced a calcium‐dependent increase in current amplitude blocked by chelerythrine. By contrast, the diacylglycerol analogue 1,2‐dioctanoyl‐sn‐glycerol produced a calcium‐independent reduction of the nicotinic response, reversed by rottlerin and chelerythrine. This indicated that two protein kinase C isozymes (‘classical’ and ‘novel’ protein kinase C, named PKC1 and PKC2, respectively) up‐ and down‐regulated nicotinic acetylcholine receptor function. PMA and 1,2‐dioctanoyl‐sn‐glycerol effects were mimicked by pirenzepine‐sensitive M1 muscarinic receptor subtype coupled to phospholipase C second messenger pathway. Low concentration of muscarine elevated internal calcium levels, which thereby activated PKC1. By contrast, a high concentration of muscarine strongly increased [Ca 2+]i, which induced inhibition of PKC1. This effect was reversed by FK506, suggesting the implication of PP2B which unmasked PKC2 activity mediating down‐regulation of nicotinic acetylcholine receptor.

Poneratoxin, a novel peptide neurotoxin from the venom of the ant, Paraponera clavata.

1. At concentrations varying from 10(-8) to 10(-6) M synthetic poneratoxin (PoTX) is a strong, but very slowly acting agonist for smooth muscles and its blocks synaptic transmission in the insect CNS in a concentration-dependent manner and depolarizes giant interneurons. 2. However, in isolated dorsal unpaired median cells 10(-6) M PoTX causes only a reversible hyperpolarization of about 5 mV. 3. At concentrations from 10(-8) to 10(-6) M PoTX affects the electrical activity of isolated cockroach axons, as well as isolated frog and rat skeletal muscle fibres. 4. PoTX prolongs action potentials and induces slow automatic activity, due to a slow Na(+)-current activation at very negative values of potential and due to slow deactivation.

Co-existence in DUM neurones of two GluCl channels that differ in their picrotoxin sensitivity

Whole cell patch-clamp electrophysiology was used to study the effects of L-glutamate on dissociated cockroach (Periplaneta americana) dorsal unpaired median (DUM) neurones. Application of L-glutamate via pressure-ejection pipette resulted in a two-component hyperpolarization, consisting of an initial transient and a second, prolonged phase. Under voltage-clamp, using isotonic chloride in the saline and intrapipette solutions, two L-glutamate-gated inward currents were characterized. Their reversal potentials were close to the equilibrium potential for chloride ions. One component was selectively activated by ibotenate and was sensitive to picrotoxin (100 μM), BIDN (10 μM) and the phenylpyrazole insecticide fipronil (10 μM), known to be potent blockers of insect GABA-gated chloride channels. The second component was insensitive to picrotoxin (100 μM) and BIDN (10 μM). These findings demonstrated, for the first time, the co-existence of two pharmacologically distinct native insect neuronal L-glutamate-gated chloride channels.

M2-like presynaptic receptors modulate acetylcholine release in the cockroach (Periplaneta americana) central nervous system

Abstract The effects of muscarinic agonists on the acetylcholine release in the cercal-afferent giant-interneurone synapses of the cockroach ( Periplaneta americana ) were investigated using the single-fibre oil-gap method. Pressure-ejection of arecoline, carbachol and oxotremorine within the sixth abdominal ganglion reduced the cEPSP amplitude in a concentration-dependent manner without any effect at the postsynaptic site in the range of concentrations used. A maximal inhibitory effect of 56, 54 and 68% was estimated for arecoline, carbachol and oxotremorine, respectively. The rank order of potencies for the agonists tested was: arecoline > carbachol > oxotremorine. McN-A-343 and bethanechol had an inhibitory effect on acetylcholine release but higher doses also acted at the postsynaptic level. The depressive effect of arecoline was only counteracted by AF-DX 116 and methoctramine. In presence of picrotoxin the inhibitory effect of arecoline and oxotremorine was reduced by about 15%. It is concluded that acetylcholine release in the cockroach central nervous system is modulated by functional M 2 -like presynaptic receptors. The influence of presynaptic electrical activity on this modulatory effect is discussed.

Anatomy and targets of dorsal unpaired median neurones in the terminal abdominal ganglion of the male cockroach Periplaneta americana L.

The morphology of the Dorsal Unpaired Median (DUM) neurones in the Terminal Abdominal Ganglion (TAG) of the adult male cockroach Periplaneta americana were described based on wholemount preparations and paraffin sections and by using anterograde and retrograde cobalt mapping, octopamine‐like immunohistochemistry, and double immunofluorescence technique with both conjugated gamma‐aminobutyric acid (GABA) and octopamine antisera. Among 60 ± 6 neurones with large somata (diameter 40 to 60 μm) on the dorsal midline surface of the TAG that were stained with toluidine blue, about 36 efferent DUM neurones exhibited octopamine‐like immunoreactivity. The DUM neurones were arranged in three clusters (anterior, median and posterior) corresponding to the 7th–11th abdominal ganglia of the fused TAG. Anterior efferent DUM neurones with one, two, and four pairs of lateral neurites entered segmental nerves VIIB; VIIB and phallic nerves; IXB and phallic nerves; VIIIA, IXA, X, and IX, respectively. Three octopamine‐like immunoreactive DUM neurones innervating heart chambers via segmental nerves (VIIA, VIIIA, and IXA) in the last abdominal segments occurred within abdominal ganglia 7, 8, and 9. Together with octopamine‐like immunoreactive efferent DUM neurones, GABA‐like immunoreactive dorsal midline neurones with small somata (10 to 20 μm) also occurred within the median group. The spatial distribution of DUM neurones in the TAG suggested that they had their origins in the median neuroblast, as for DUM neurones in the grasshopper.

Refined electrophysiological analysis suggests that a depressant toxin is a sodium channel opener rather than a blocker.

The effects of a recombinant depressant insect toxin from Leiurus quinquestriatus hebraeus, Lqh IT2-r, have been studied in current and voltage-clamp conditions on the isolated axonal and DUM neuron preparations of the cockroach Periplaneta americana. Lqh IT2-r depolarizes the axon, blocks the evoked action potentials, and modifies the amplitude and the kinetics of the sodium current. The inward transient peak current is greatly decreased and is followed by a maintained slow activating-deactivating sodium current. The slow component develops at membrane potentials more negative than the control, and has a time constant of activation of several tens of milliseconds. The flaccid properties of Lqh IT2-r do not correspond to a blockage of the Na+ channels, but may be attributed to modified Na+ channels which open at more negative potential, activate slowly and do not inactivate normally.