Le Mauff A et al. (2020), Nicotinic acetylcholine receptors in the syngan...

XB-ART-57538

Int J Parasitol Drugs Drug Resist 2020 Dec 01;14:144-151. doi: 10.1016/j.ijpddr.2020.10.005.

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

Le Mauff A , Chouikh H , Cartereau A , Charvet CL , Neveu C , Rispe C , Plantard O , Taillebois E , Thany SH .

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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.

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Species referenced: Xenopus laevis
Genes referenced: ace

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	Fig. 1. Properties of acetylcholine and nicotine currents in oocytes microtransplanted with tick synganglion membranes. (A and B) Currents elicited by 1 mM acetylcholine (ACh). Top current without atropine (control, Ctl) and below with 0.5 μM atropine (Atr). Horizontal bars above each response indicate the compound application. The perfusion time was 20 s. Histograms illustrate the comparison between ACh without atropine (Ctl) or with 0.5 μM atropine (Atr) (p > 0.05, n = 16, ns = no significant). (C and D) Currents elicited by 1 mM nicotine (Nic) compared in control condition (Ctl: without atropine) and under bath application of 0.5 μM atropine (Atr). Histograms show that there are no significant changes in the current amplitude (p > 0.05, n = 23, ns = no significant). (E) ACh and nicotine concentration-current response relationships from n = 12, in the presence of 0.5 μM atropine. Each point represents peak currents normalized to Imean. (F and G) Currents elicited by 1 mM muscarine. Top current represents 1 mM muscarine (Mus)-induced current, and below the blocking activity induced by 0.5 μM atropine. Histograms illustrate the current amplitudes between muscarine without atropine (control condition, Ctl) and with 0.5 μM atropine (Atr). n = 8, *p < 0.05. In each histogram, data are mean ± S.E.M.
	Fig. 2. Effect of α-Bgt on ACh- and nicotine-evoked currents in oocyte microtransplanted with tick synganglion membranes. (A and B) Currents elicited by 1 mM ACh. Top current without α-Bgt and below with 10 nM α-Bgt. Horizontal bars above each response indicate the compound application, and the perfusion time was 20 s. Histograms illustrate the comparison between ACh without 10 nM α-Bgt (Ctl) or with 10 nM α-Bgt. P > 0.05, n = 9, ns = no significant. (C and D) Currents elicited by 1 mM nicotine (Nic), without and under bath application of 10 nM α-Bgt. Histograms illustrating current amplitudes when α-Bgt is added in the bath. Each histogram represents n = 10, *p < 0.05.
	Fig. 3. Fluorescent labelling of tick synganglion membranes showing the presence of nAChRs α-Bgt -sensitive subtypes, using fluorescence microscopy. (A) Synganglion membrane stained with Alexa Fluor 488-α-Bgt complex. (B) The same preparation as A, visualized with transmitted light. (C) Synganglion membrane as a control preparation, without Alexa Fluor 488-α-Bgt complex. (D) The same preparation as C, visualized with transmitted light. Scale bar = 200 μm.
	Fig. 4. Modulatory effect of PNU-120596 (PNU) on ACh- and nicotine-evoked currents in oocytes microtransplanted with tick synganglion membranes. (A and B) Currents elicited by 1 mM ACh. Top current without PNU-120596 (PNU) and below with 1 μM PNU-120596. Each horizontal bars indicates when the compound us added. Histograms illustrate the comparison between ACh without PNU-120596 (Ctl) or with 1 μM PNU-120596. P > 0.05, n = 8, ns = no significant. (C and D) Currents elicited by 1 mM nicotine and under bath application of 1 μM PNU-120596. Histograms show that there is no significant difference in the current amplitude when PNU-120596 was added in the bath. P > 0,05, n = 9, ns = no significant. Each histogram represents mean ± S.E.M, and the perfusion time was 20 s.
	Fig. 5. Modulatory effect of combined application of atropine and PNU-120596 on ACh- and nicotine-evoked currents in oocyte microtransplanted with tick synganglion membranes. (A and B) In top: currents elicited by 1 mM ACh in presence of 0.5 μM atropine (Atr). Below: in the presence of 0.5 μM atropine (Atr) and 1 μM PNU-120596 (PNU). Horizontal bars indicate the compound application. Histograms illustrate the mean current amplitudes and represent, n = 11. (C and D) Currents induced by 1 mM nicotine under 0.5 μM atropine (Atr) and bath application of 0.5 μM atropine and 1 μM PNU (PNU). Histograms illustrate current amplitudes and show that there is a significant difference. Each histogram represents n = 23. Note that in all case, the perfusion time for ACh and nicotine was 20 s, histogram are mean ± S.E.M and *p < 0.05.
	Fig. 6. Effect of α-Bgt under bath application of 0.5 μM atropine (Atr) and 1 μM PNU-120596. (A and B) Currents elicited by 1 mM ACh without 10 nM α-Bgt, top current, and with 10 nM α-Bgt (below). Horizontal bars indicate the compound application. Histograms illustrate currents recorded in the same conditions, and represent n = 7. (C and D) Currents elicited by 1 mM nicotine (Nic) without α-Bgt and under bath application of 10 nM α-Bgt. Histograms show that there is a significant difference in the current amplitude when 10 nM α-Bgt is added in the bath. Each histograms represent n = 13. In all case, each histogram represent mean ± S.E.M and * p < 0.05.
	Fig. 7. Effect of mecamylamine and methyllycaconitine on ACh-evoked currents. 1 mM ACh (20 s, horizontal bar) currents were recorded under bath application of 0.5 μM atropine (Atr) and 1 μM PNU-120596 (PNU). (A and B) As shown, 1 μM mecamylamine (Meca) has no effect on 1 mM ACh-induced currents. Each histogram represents mean ± S.E.M of n = 12. ns = no significant and p > 0.05. (C and D) Similarly, 10 nM MLA has no effect on ACh currents. Histograms illustrate the current amplitudes and represents mean ± S.E.M of n = 7, p > 0.05. Ctl: represents application of ACh without 1 μM mecamylamine or 10 nM MLA.
	Fig. 8. Effect of mecamylamine and methyllycaconitine on nicotine-evoked currents. All currents were recorded under bath application of 0.5 μM atropine (Atr) and 1 μM PNU-120596 (PNU). (A and B) Currents elicited by 1 mM nicotine (Nic) are not affected by 1 μM mecamylamine (Meca). Each histogram represents mean ± S.E.M of n = 9, ns = no significant, p > 0.05. (C and D) Currents elicited by 1 mM nicotine (Nic, 20 s, horizontal bar) are diminished by 10 nM MLA. Histograms illustrate the effect of 10 nM MLA, and represent mean ± S.E.M of n = 12, *p < 0.05. Ctl: represents application of 1 mM nicotine without 1 μM mecamylamine or 10 nM MLA.
	Fig. 9. Effect of PNU-120596 on current evoked by the neonicotinoid compounds, clothianidin (CLT), imidacloprid (IMI), thiamethoxam (TMX) and acetamiprid (ACE), in oocyte microtransplanted with tick synganglion membranes. In all case, we found that, current induced by 1 mM of each neonicotinoid are significantly increased under bath application of 1 μM PNU-120596. (A and B) Currents and histograms under 1 mM CLT (n = 9, p < 0.05). (C and D) Currents induced by 1 mM IMI and the corresponding histograms (n = 19, p < 0.05). (E and F) Currents and histograms representing currents elicited by 1 mM TMX (n = 14, p < 0.05). (G and H) Currents elicited by 1 mM ACE and Histograms illustrating the increase under bath application of PNU-120596 (n = 20, p < 0.05). In all case, histograms are mean ± S.E.M. All compounds were applied during 20 s (black bars). Ctl: represents application of each compound without 1 μM PNU in the bath.

References [+] :

Barbour, Discovery of the Lyme Disease Agent. 2019, Pubmed

Barbour, Discovery of the Lyme Disease Agent. 2019, Pubmed
Bissinger, Synganglion transcriptome and developmental global gene expression in adult females of the American dog tick, Dermacentor variabilis (Acari: Ixodidae). 2011, Pubmed
Cardenas-de la Garza, Clinical spectrum of Lyme disease. 2019, Pubmed
Cartereau, Neonicotinoid insecticides differently modulate acetycholine-induced currents on mammalian α7 nicotinic acetylcholine receptors. 2018, Pubmed , Xenbase
Collin, Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods. 2013, Pubmed
Courjaret, Two distinct calcium-sensitive and -insensitive PKC up- and down-regulate an alpha-bungarotoxin-resistant nAChR1 in insect neurosecretory cells (DUM neurons). 2003, Pubmed
Crespin, Injection of insect membrane in Xenopus oocyte: An original method for the pharmacological characterization of neonicotinoid insecticides. 2016, Pubmed , Xenbase
Davies, Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling alpha 7-type neuronal nicotinic acetylcholine receptors. 1999, Pubmed
Egekwu, Transcriptome of the female synganglion of the black-legged tick Ixodes scapularis (Acari: Ixodidae) with comparison between Illumina and 454 systems. 2014, Pubmed
Eusebi, Microtransplantation of ligand-gated receptor-channels from fresh or frozen nervous tissue into Xenopus oocytes: a potent tool for expanding functional information. 2009, Pubmed , Xenbase
Gulia-Nuss, Genomic insights into the Ixodes scapularis tick vector of Lyme disease. 2016, Pubmed , Xenbase
Hammond, The bradford method for protein quantitation. 1988, Pubmed
Houchat, Mode of action of sulfoxaflor on α-bungarotoxin-insensitive nAChR1 and nAChR2 subtypes: Inhibitory effect of imidacloprid. 2019, Pubmed
Jongejan, The global importance of ticks. 2004, Pubmed
Kalappa, The dual effect of PNU-120596 on α7 nicotinic acetylcholine receptor channels. 2013, Pubmed
Lees, Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus. 2014, Pubmed , Xenbase
Lees, Transcriptome analysis of the synganglion from the brown dog tick, Rhipicephalus sanguineus. 2010, Pubmed
Lindquist, Tick-borne encephalitis (TBE) in childhood. 2008, Pubmed
Marsal, Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes. 1995, Pubmed , Xenbase
Miledi, Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes. 2002, Pubmed , Xenbase
Millar, Diversity of vertebrate nicotinic acetylcholine receptors. 2009, Pubmed
Murenzi, Evaluation of microtransplantation of rat brain neurolemma into Xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage-sensitive ion channels. 2017, Pubmed , Xenbase
Palma, Microtransplantation of membranes from cultured cells to Xenopus oocytes: a method to study neurotransmitter receptors embedded in native lipids. 2003, Pubmed , Xenbase
Taillebois, Neonicotinoid insecticides mode of action on insect nicotinic acetylcholine receptors using binding studies. 2018, Pubmed
Tan, Agonist actions of neonicotinoids on nicotinic acetylcholine receptors expressed by cockroach neurons. 2007, Pubmed
Thany, Agonist actions of clothianidin on synaptic and extrasynaptic nicotinic acetylcholine receptors expressed on cockroach sixth abdominal ganglion. 2009, Pubmed
Thany, Exploring the pharmacological properties of insect nicotinic acetylcholine receptors. 2007, Pubmed
Thany, Thiamethoxam, a poor agonist of nicotinic acetylcholine receptors expressed on isolated cell bodies, acts as a full agonist at cockroach cercal afferent/giant interneuron synapses. 2011, Pubmed
Tomizawa, The neonicotinoid electronegative pharmacophore plays the crucial role in the high affinity and selectivity for the Drosophila nicotinic receptor: an anomaly for the nicotinoid cation--pi interaction model. 2003, Pubmed