Tetrodotoxin Decreases the Contractility of Mesenteric Arteries, Revealing the Contribution of Voltage-Gated Na+ Channels in Vascular Tone Regulation.

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na+ (NaV) channels. In arteries, NaV channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of NaV channels in vascular tone using TTX. We first characterized the expression of NaV channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular NaV channels consist of NaV1.2 channel subtype with NaVß1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks NaV channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications.

The multiple facets of mitochondrial regulations controlling cellular thermogenesis.

Understanding temperature production and regulation in endotherm organisms becomes a crucial challenge facing the increased frequency and intensity of heat strokes related to global warming. Mitochondria, located at the crossroad of metabolism, respiration, Ca2+ homeostasis, and apoptosis, were recently proposed to further act as cellular radiators, with an estimated inner temperature reaching 50 °C in common cell lines. This inner thermogenesis might be further exacerbated in organs devoted to produce consistent efforts as muscles, or heat as brown adipose tissue, in response to acute solicitations. Consequently, pathways promoting respiratory chain uncoupling and mitochondrial activity, such as Ca2+ fluxes, uncoupling proteins, futile cycling, and substrate supplies, provide the main processes controlling heat production and cell temperature. The mitochondrial thermogenesis might be further amplified by cytoplasmic mechanisms promoting the over-consumption of ATP pools. Considering these new thermic paradigms, we discuss here all conventional wisdoms linking mitochondrial functions to cellular thermogenesis in different physiological conditions.

Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells.

Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the ß-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

GABAA Receptor Subunit Composition Drives Its Sensitivity to the Insecticide Fipronil.

Fipronil (FPN) is a worldwide-used neurotoxic insecticide, targeting, and blocking GABAA receptors (GABAARs). Beyond its efficiency on insect GABAARs, FPN causes neurotoxic effects in humans and mammals. Here, we investigated the mode of action of FPN on mammalian α6-containing GABAARs to understand its inhibitory effects on GABA-induced currents, as a function of the synaptic or extrasynaptic localization of GABAARs. We characterized the effects of FPN by electrophysiology using Xenopus oocytes which were microtransplanted with cerebellum membranes or injected with α6ß3, α6ß3γ2S (synaptic), and α6ß3δ (extrasynaptic) cDNAs. At micromolar concentrations, FPN dose-dependently inhibited cerebellar GABA currents. FPN acts as a non-competitive antagonist on ternary receptors. Surprisingly, the inhibition of GABA-induced currents was partial for extra-synaptic (α6ß3δ) and binary (α6ß3) receptors, while synaptic α6ß3γ2S receptors were fully blocked, indicating that the complementary γ or δ subunit participates in FPN-GABAAR interaction. FPN unexpectedly behaved as a positive modulator on ß3 homopentamers. These data show that FPN action is driven by the subunit composition of GABAARs-highlighting the role of the complementary subunit-and thus their localization within a physiological synapse. We built a docking model of FPN on GABAARs, which reveals two putative binding sites. This is consistent with a double binding mode of FPN on GABAARs, possibly one being of high affinity and the other of low affinity. Physiologically, the γ/δ subunit incorporation drives its inhibitory level and has important significance for its toxicity on the mammalian nervous system, especially in acute exposure.

Partial Agonist Activity of Neonicotinoids on Rat Nicotinic Receptors: Consequences over Epinephrine Secretion and In Vivo Blood Pressure.

Neonicotinoid insecticides are nicotine-derived molecules which exert acute neurotoxic effects over the insect central nervous system by activating nicotinic acetylcholine receptors (nAChRs). However, these receptors are also present in the mammalian central and peripheral nervous system, where the effects of neonicotinoids are faintly known. In mammals, cholinergic synapses are crucial for the control of vascular tone, blood pressure and skeletal muscle contraction. We therefore hypothesized that neonicotinoids could affect cholinergic networks in mammals and sought to highlight functional consequences of acute intoxication in rats with sub-lethal concentrations of the highly used acetamiprid (ACE) and clothianidin (CLO). In this view, we characterized their electrophysiological effects on rat α3ß4 nAChRs, knowing that it is predominantly expressed in ganglia of the vegetative nervous system and the adrenal medulla, which initiates catecholamine secretion. Both molecules exhibited a weak agonist effect on α3ß4 receptors. Accordingly, their influence on epinephrine secretion from rat adrenal glands was also weak at 100 µM, but it was stronger at 500 µM. Challenging ACE or CLO together with nicotine (NIC) ended up with paradoxical effects on secretion. In addition, we measured the rat arterial blood pressure (ABP) in vivo by arterial catheterization. As expected, NIC induced a significant increase in ABP. ACE and CLO did not affect the ABP in the same conditions. However, simultaneous exposure of rats to both NIC and ACE/CLO promoted an increase of ABP and induced a biphasic response. Modeling the interaction of ACE or CLO on α3ß4 nAChR is consistent with a binding site located in the agonist pocket of the receptor. We present a transversal experimental approach of mammal intoxication with neonicotinoids at different scales, including in vitro, ex vivo, in vivo and in silico. It paves the way of the acute and chronic toxicity for this class of insecticides on mammalian organisms.

Anatoxin-a: Overview on a harmful cyanobacterial neurotoxin from the environmental scale to the molecular target.

Anatoxin-a (ATX-a) is a neurotoxic alkaloid, produced by several freshwater planktonic and benthic cyanobacteria (CB). Such CB have posed human and animal health issues for several years, as this toxin is able to cause neurologic symptoms in humans following food poisoning and death in wild and domestic animals. Different episodes of animal intoxication have incriminated ATX-a worldwide, as confirmed by the presence of ATX-a-producing CB in the consumed water or biofilm, or the observation of neurotoxic symptoms, which match experimental toxicity in vivo. Regarding toxicity parameters, toxicokinetics knowledge is currently incomplete and needs to be improved. The toxin can passively cross biological membranes and act rapidly on nicotinic receptors, its main molecular target. In vivo and in vitro acute effects of ATX-a have been studied and make possible to draw its mode of action, highlighting its deleterious effects on the nervous systems and its effectors, namely muscles, heart and vessels, and the respiratory apparatus. However, very little is known about its putative chronic toxicity. This review updates available data on ATX-a, from the ecodynamic of the toxin to its physiological and molecular targets.

An Insecticide Further Enhances Experience-Dependent Increased Behavioural Responses to Sex Pheromone in a Pest Insect.

Neonicotinoid insecticides are widely used to protect plants against pest insects, and insecticide residues remaining in the environment affect both target and non-target organisms. Whereas low doses of neonicotinoids have been shown to disturb the behaviour of pollinating insects, recent studies have revealed that a low dose of the neonicotinoid clothianidin can improve behavioural and neuronal sex pheromone responses in a pest insect, the male moth Agrotis ipsilon, and thus potentially improve reproduction. As male moth behaviour depends also on its physiological state and previous experience with sensory signals, we wondered if insecticide effects would be dependent on plasticity of olfactory-guided behaviour. We investigated, using wind tunnel experiments, whether a brief pre-exposure to the sex pheromone could enhance the behavioural response to this important signal in the moth A. ipsilon at different ages (sexually immature and mature males) and after different delays (2 h and 24 h), and if the insecticide clothianidin would interfere with age effects or the potential pre-exposure-effects. Brief pre-exposure to the pheromone induced an age-independent significant increase of sex pheromone responses 24 h later, whereas sex pheromone responses did not increase significantly 2 h after exposure. However, response delays were significantly shorter compared to naïve males already two hours after exposure. Oral treatment with clothianidin increased sex pheromone responses in sexually mature males, confirming previous results, but did not influence responses in young immature males. Males treated with clothianidin after pre-exposure at day 4 responded significantly more to the sex pheromone at day 5 than males treated with clothianidin only and than males pre-exposed only, revealing an additive effect of experience and the insecticide. Plasticity of sensory systems has thus to be taken into account when investigating the effects of sublethal doses of insecticides on behaviour.

Low doses of a neonicotinoid insecticide modify pheromone response thresholds of central but not peripheral olfactory neurons in a pest insect.

Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids, leaving residues in the environment. There is now evidence that low doses of insecticides can have positive effects on pest insects by enhancing various life traits. Because pest insects often rely on sex pheromones for reproduction, and olfactory synaptic transmission is cholinergic, neonicotinoid residues could modify chemical communication. We recently showed that treatments with different sublethal doses of clothianidin could either enhance or decrease behavioural sex pheromone responses in the male moth, Agrotis ipsilon. We investigated now effects of the behaviourally active clothianidin doses on the sensitivity of the peripheral and central olfactory system. We show with extracellular recordings that both tested clothianidin doses do not influence pheromone responses in olfactory receptor neurons. Similarly, in vivo optical imaging does not reveal any changes in glomerular response intensities to the sex pheromone after clothianidin treatments. The sensitivity of intracellularly recorded antennal lobe output neurons, however, is upregulated by a lethal dose 20 times and downregulated by a dose 10 times lower than the lethal dose 0. This correlates with the changes of behavioural responses after clothianidin treatment and suggests the antennal lobe as neural substrate involved in clothianidin-induced behavioural changes.

Injection of insect membrane in Xenopus oocyte: An original method for the pharmacological characterization of neonicotinoid insecticides.

INTRODUCTION: Insect nicotinic acetylcholine receptors (nAChRs) represent a major target of insecticides, belonging to the neonicotinoid family. However, the pharmacological profile of native nAChRs is poorly documented, mainly because of a lack of knowledge of their subunit stoichiometry, their tissue distribution and the weak access to nAChR-expressing cells. In addition, the expression of insect nAChRs in heterologous systems remains hard to achieve. Therefore, the structure-activity characterization of nAChR-targeting insecticides is made difficult. The objective of the present study was to characterize insect nAChRs by an electrophysiological approach in a heterologous system naturally devoid of these receptors to allow a molecular/cellular investigation of the mode of action of neonicotinoids. Methods To overcome impediments linked to the expression of insect nAChR mRNA or cDNA, we chose to inject insect membranes from the pea aphid (Acyrthosiphon pisum) into Xenopus oocytes. This microtransplantation technique was designed to gain access to native nAChRs embedded in their membrane, through direct stimulation with nicotinic agonists. Results We provide evidence that an enriched-nAChR membrane allows us to characterize native receptors. The presence of such receptors was confirmed with fluorescent α-BgTX labeling. Electrophysiological recordings of nicotine-induced inward currents allowed us to challenge the presence of functional nAChR. We compared the effect of nicotine (NIC) with clothianidin (CLO) and we assessed the effect of thiamethoxam (TMX). Discussion This technique has been recently highlighted with mammalian and human material as a powerful functional approach, but has, to our knowledge, never been used with insect membrane. In addition, the use of the insect membrane microtransplantation opens a new and original way for pharmacological screening of neurotoxic insecticides, including neonicotinoids. Moreover, it might also be a powerful tool to investigate the pharmacological properties of insect nAChR.

Unexpected effects of low doses of a neonicotinoid insecticide on behavioral responses to sex pheromone in a pest insect.

In moths, which include many agricultural pest species, males are attracted by female-emitted sex pheromones. Although integrated pest management strategies are increasingly developed, most insect pest treatments rely on widespread use of neurotoxic chemicals, including neonicotinoid insecticides. Residual accumulation of low concentrations of these insecticides in the environment is known to be harmful to beneficial insects such as honey bees. This environmental stress probably acts as an "info-disruptor" by modifying the chemical communication system, and therefore decreases chances of reproduction in target insects that largely rely on olfactory communication. However, low doses of pollutants could on the contrary induce adaptive processes in the olfactory pathway, thus enhancing reproduction. Here we tested the effects of acute oral treatments with different low doses of the neonicotinoid clothianidin on the behavioral responses to sex pheromone in the moth Agrotis ipsilon using wind tunnel experiments. We show that low doses of clothianidin induce a biphasic effect on pheromone-guided behavior. Surprisingly, we found a hormetic-like effect, improving orientation behavior at the LD20 dose corresponding to 10 ng clothianidin. On the contrary, a negative effect, disturbing orientation behavior, was elicited by a treatment with a dose below the LD0 dose corresponding to 0.25 ng clothianidin. No clothianidin effect was observed on behavioral responses to plant odor. Our results indicate that risk assessment has to include unexpected effects of residues on the life history traits of pest insects, which could then lead to their adaptation to environmental stress.

Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum.

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM) and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml) and TMX (LC50 = 0.034 µg/ml) were more toxic than CLT (LC50 = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumß1 expressions levels, whereas Apisumß2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.

Ca²âº/calmodulin-dependent protein kinase II in the cockroach Periplaneta americana: identification of five isoforms and their tissues distribution.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key kinase that transduces Ca²âº signals into downstream effects acting on a range of cellular processes in nervous system and muscular tissues. In insects, different CaMKII isoforms have been reported in Drosophila melanogaster, Apis florae, Bombus terrestris, and Bombus impatiens but little is known on the organization and tissue-specific expression of these isoforms with the exception of Drosophila. The present study reports the cloning of five CaMKII splice variants issued from a single gene and their tissue-specific expression in the cockroach Periplaneta americana. Each CaMKII isoform shared 82-90% identity with Drosophila CaMKII isoforms and accordingly were named PaCaMKII-A, PaCaMKII-B,PaCaMKII-C,PaCaMKII-D, and PaCaMKII-E. PaCaMKII-A and PaCaMKII-D isoforms are ubiquitously expressed in all tissues, but some such as PaCaMKII-B andPaCaMKII-C are preferentially expressed in the nerve cord and muscle. In addition, using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we found a tissue-specific expression of PaCaMKII-E in the dorsal unpaired median neurons. Alternative splicing of PaCaMKII transcripts is likely a common mechanism in insects to control the pattern of isoform expression in the different tissues.

Pest insect olfaction in an insecticide-contaminated environment: info-disruption or hormesis effect.

Most animals, including pest insects, live in an "odor world" and depend strongly on chemical stimuli to get information on their biotic and abiotic environment. Although integrated pest management strategies including the use of insect growth regulators (IGRs) are increasingly developed, most insect pest treatments rely on neurotoxic chemicals. These molecules are known to disrupt synaptic transmission, affecting therefore sensory systems. The wide-spread use of neurotoxic insecticides and the growing use of IGRs result in residual accumulation of low concentrations in the environment. These insecticide residues could act as an "info-disruptor" by modifying the chemical communication system, and therefore decrease chances of reproduction in target insects. However, residues can also induce a non-expected hormesis effect by enhancing reproduction abilities. Low insecticide doses might thus induce adaptive processes in the olfactory pathway of target insects, favoring the development of resistance. The effect of sublethal doses of insecticides has mainly been studied in beneficial insects such as honeybees. We review here what is known on the effects of sublethal doses of insecticides on the olfactory system of insect pests.

Emerging Pharmacological Properties of Cholinergic Synaptic Transmission: Comparison between Mammalian and Insect Synaptic and Extrasynaptic Nicotinic Receptors.

Acetylcholine (ACh) is probably the oldest signalling neurotransmitter which appeared in evolution before the nervous system. It is present in bacteria, algae, protozoa and plants. In insects and mammals it is involved in cell-to-cell communications in various neuronal and non-neuronal tissues. The discovery of nicotinic acetylcholine receptors (nAChRs) as the main receptors involved in rapid cholinergic neurotransmission has helped to understand the role of ACh at synaptic level. Recently, several lines of evidence have indicated that extrasynaptically expressed nAChRs display distinct pharmacological properties from the ones expressed at synaptic level. The role of both nAChRs at insect extrasynaptic and/or synaptic levels has been underestimated due to the lack of pharmacological tools to identify different nicotinic receptor subtypes. In the present review, we summarize recent electrophysiological and pharmacological studies on the extrasynaptic and synaptic differences between insect and mammalian nAChR subtypes and we discuss on the pharmacological impact of several drugs such as neonicotinoid insecticides targeting these receptors. In fact, nAChRs are involved in a wide range of pathophysiological processes such as epilepsy, pain and a wide range of neurodegenerative and psychiatric disorders. In addition, they are the target sites of neonicotinoid insecticides which are known to act as nicotinic agonists causing severe poisoning in insects and mammals.

Identification of cholinergic synaptic transmission in the insect nervous system.

A major criteria initially used to localize cholinergic neuronal elements in nervous systems tissues that involve acetylcholine (ACh) as neurotransmitter is mainly based on immunochemical studies using choline acetyltransferase (ChAT), an enzyme which catalyzes ACh biosynthesis and the ACh degradative enzyme named acetylcholinesterase (AChE). Immunochemical studies using anti-ChAT monoclonal antibody have allowed the identification of neuronal processes and few types of cell somata that contain ChAT protein. In situ hybridization using cRNA probes to ChAT or AChE messenger RNA have brought new approaches to further identify cell bodies transcribing the ChAT or AChE genes. Combined application of all these techniques reveals a widespread expression of ChAT and AChE activities in the insect central nervous system and peripheral sensory neurons which implicates ACh as a key neurotransmitter. The discovery of the snake toxin alpha-bungatoxin has helped to identify nicotinic acetylcholine receptors (nAChRs). In fact, nicotine when applied to insect neurons, resulted in the generation of an inward current through the activation of nicotinic receptors which were blocked by alpha-bungarotoxin. Thus, insect nAChRs have been divided into two categories, sensitive and insensitive to this snake toxin. Up to now, the recent characterization and distribution pattern of insect nAChR subunits and the biochemical evidence that the insect central nervous system contains different classes of cholinergic receptors indicated that ACh is involved in several sensory pathways.

Identification of critical elements determining toxins and insecticide affinity, ligand binding domains and channel properties.

Insect nicotinic acetylcholine receptors have been objects of attention since the discovery of neonicotinoid insecticides. Mutagenesis studies have revealed that, although the detailed subunit composition of insect nicotinic acetylcholine receptors subtypes eludes us, the framework provided by mutagenesis analysis makes a picture of the subunits involved in the ligand binding and channel properties. In fact, many residues that line the channel or bind to the ligand seemed to be strongly conserved in particular in the N-terminal extracellular region and the second transmembrane domain which constitutes the ion-conducting pathway supporting the flux of ions as well as their discrimination. In fact, the positions are carried by loops B and C, respectively, which contain amino acids directly contributing to the acetylcholine binding site. Mutation ofthese residues accounts for insect resistance to neonicotinoid insecticides such as imidacloprid or a loss ofspecific binding. The discovery of the same mutation at homologous residues in different insect species or its conservation raises the intriguing question of whether a single mutation is essential to generate a resistance phenotype or whether some subunit confer insensitivity to ligand. Consequently, recent finding using information from Torpedo marmorata al subunit and soluble Aplysia californica and Lymnae stagnalis acetylcholine bindingproteins from crystallization suggest that insect nAChR subunits had contributing amino acids in the agonist site structure which participate to affinity and pharmacological properties of these receptors. These new range of data greatly facilitate the understanding of toxin-nAChR interactions and the neonicotinoid binding and selectivity.

Molecular mechanism of systemic delivery of neural precursor cells to the brain: assembly of brain endothelial apical cups and control of transmigration by CD44.

Systemically injected neural precursor cells (NPCs) were unexpectedly shown to reach the cerebral parenchyma and induce recovery in various diffuse brain pathologies, including animal models of multiple sclerosis. However, the molecular mechanisms supporting NPC migration across brain endothelium remain elusive. Brain endothelium constitutes the blood-brain barrier, which uniquely controls the access of drugs and trafficking of cells, including leukocytes, from the blood to the brain. Taking advantage of the availability of in vitro models of human and rat blood-brain barrier developed in our laboratory and validated by us and others, we show here that soluble hyaluronic acid, the major ligand of the adhesion molecule CD44, as well as anti-CD44 blocking antibodies, largely prevents NPC adhesion to and migration across brain endothelium in inflammatory conditions. We present further evidence that NPCs, surprisingly, induce the formation of apical cups at the surface of brain endothelial cells, enriched in CD44 and other adhesion molecules, thus hijacking the endothelial signaling recently shown to be involved in leukocyte extravasation. These results demonstrate the pivotal role of CD44 in the trans-endothelial migration of NPCs across brain endothelial cells: we propose that they may help design new strategies for the delivery of therapeutic NPCs to the brain by systemic administration.