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 1Â mM acetylcholine (ACh). Top current without atropine (control, Ctl) and below with 0.5Â Î¼M atropine (Atr). Horizontal bars above each response indicate the compound application. The perfusion time was 20Â s. Histograms illustrate the comparison between ACh without atropine (Ctl) or with 0.5Â Î¼M atropine (Atr) (pÂ >Â 0.05, nÂ =Â 16, nsÂ =Â no significant). (C and D) Currents elicited by 1Â mM nicotine (Nic) compared in control condition (Ctl: without atropine) and under bath application of 0.5Â Î¼M atropine (Atr). Histograms show that there are no significant changes in the current amplitude (pÂ >Â 0.05, nÂ =Â 23, nsÂ =Â no significant). (E) ACh and nicotine concentration-current response relationships from nÂ =Â 12, in the presence of 0.5Â Î¼M atropine. Each point represents peak currents normalized to Imean. (F and G) Currents elicited by 1Â mM muscarine. Top current represents 1Â mM muscarine (Mus)-induced current, and below the blocking activity induced by 0.5Â Î¼M atropine. Histograms illustrate the current amplitudes between muscarine without atropine (control condition, Ctl) and with 0.5Â Î¼M atropine (Atr). nÂ =Â 8, *pÂ <Â 0.05. In each histogram, data are meanÂ Â±Â S.E.M.
	Fig. 2. Effect of Î±-Bgt on ACh- and nicotine-evoked currents in oocyte microtransplanted with tick synganglion membranes. (A and B) Currents elicited by 1Â mM ACh. Top current without Î±-Bgt and below with 10Â nM Î±-Bgt. Horizontal bars above each response indicate the compound application, and the perfusion time was 20Â s. Histograms illustrate the comparison between ACh without 10Â nM Î±-Bgt (Ctl) or with 10Â nM Î±-Bgt. PÂ >Â 0.05, nÂ =Â 9, nsÂ =Â no significant. (C and D) Currents elicited by 1Â mM nicotine (Nic), without and under bath application of 10Â nM Î±-Bgt. Histograms illustrating current amplitudes when Î±-Bgt is added in the bath. Each histogram represents nÂ =Â 10, *pÂ <Â 0.05.
	Fig. 3. Fluorescent labelling of tick synganglion membranes showing the presence of nAChRs Î±-Bgt -sensitive subtypes, using fluorescence microscopy. (A) Synganglion membrane stained with Alexa Fluor 488-Î±-Bgt complex. (B) The same preparation as A, visualized with transmitted light. (C) Synganglion membrane as a control preparation, without Alexa Fluor 488-Î±-Bgt complex. (D) The same preparation as C, visualized with transmitted light. Scale barÂ =Â 200Â Î¼m.
	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 1Â mM ACh. Top current without PNU-120596 (PNU) and below with 1Â Î¼M PNU-120596. Each horizontal bars indicates when the compound us added. Histograms illustrate the comparison between ACh without PNU-120596 (Ctl) or with 1Â Î¼M PNU-120596. PÂ >Â 0.05, nÂ =Â 8, nsÂ =Â no significant. (C and D) Currents elicited by 1Â mM nicotine and under bath application of 1Â Î¼M PNU-120596. Histograms show that there is no significant difference in the current amplitude when PNU-120596 was added in the bath. PÂ >Â 0,05, nÂ =Â 9, nsÂ =Â no significant. Each histogram represents meanÂ Â±Â S.E.M, and the perfusion time was 20Â 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 1Â mM ACh in presence of 0.5Â Î¼M atropine (Atr). Below: in the presence of 0.5Â Î¼M atropine (Atr) and 1Â Î¼M PNU-120596 (PNU). Horizontal bars indicate the compound application. Histograms illustrate the mean current amplitudes and represent, nÂ =Â 11. (C and D) Currents induced by 1Â mM nicotine under 0.5Â Î¼M atropine (Atr) and bath application of 0.5Â Î¼M atropine and 1Â Î¼M PNU (PNU). Histograms illustrate current amplitudes and show that there is a significant difference. Each histogram represents nÂ =Â 23. Note that in all case, the perfusion time for ACh and nicotine was 20Â s, histogram are meanÂ Â±Â S.E.M and *pÂ <Â 0.05.
	Fig. 6. Effect of Î±-Bgt under bath application of 0.5Â Î¼M atropine (Atr) and 1Â Î¼M PNU-120596. (A and B) Currents elicited by 1Â mM ACh without 10Â nM Î±-Bgt, top current, and with 10Â nM Î±-Bgt (below). Horizontal bars indicate the compound application. Histograms illustrate currents recorded in the same conditions, and represent nÂ =Â 7. (C and D) Currents elicited by 1Â mM nicotine (Nic) without Î±-Bgt and under bath application of 10Â nM Î±-Bgt. Histograms show that there is a significant difference in the current amplitude when 10Â nM Î±-Bgt is added in the bath. Each histograms represent nÂ =Â 13. In all case, each histogram represent meanÂ Â±Â S.E.M and * pÂ <Â 0.05.
	Fig. 7. Effect of mecamylamine and methyllycaconitine on ACh-evoked currents. 1Â mM ACh (20Â s, horizontal bar) currents were recorded under bath application of 0.5Â Î¼M atropine (Atr) and 1Â Î¼M PNU-120596 (PNU). (A and B) As shown, 1Â Î¼M mecamylamine (Meca) has no effect on 1Â mM ACh-induced currents. Each histogram represents meanÂ Â±Â S.E.M of nÂ =Â 12. nsÂ =Â no significant and pÂ >Â 0.05. (C and D) Similarly, 10Â nM MLA has no effect on ACh currents. Histograms illustrate the current amplitudes and represents meanÂ Â±Â S.E.M of nÂ =Â 7, pÂ >Â 0.05. Ctl: represents application of ACh without 1Â Î¼M mecamylamine or 10Â nM MLA.
	Fig. 8. Effect of mecamylamine and methyllycaconitine on nicotine-evoked currents. All currents were recorded under bath application of 0.5Â Î¼M atropine (Atr) and 1Â Î¼M PNU-120596 (PNU). (A and B) Currents elicited by 1Â mM nicotine (Nic) are not affected by 1Â Î¼M mecamylamine (Meca). Each histogram represents meanÂ Â±Â S.E.M of nÂ =Â 9, nsÂ =Â no significant, pÂ >Â 0.05. (C and D) Currents elicited by 1Â mM nicotine (Nic, 20Â s, horizontal bar) are diminished by 10Â nM MLA. Histograms illustrate the effect of 10Â nM MLA, and represent meanÂ Â±Â S.E.M of nÂ =Â 12, *pÂ <Â 0.05. Ctl: represents application of 1Â mM nicotine without 1Â Î¼M mecamylamine or 10Â 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 1Â mM of each neonicotinoid are significantly increased under bath application of 1Â Î¼M PNU-120596. (A and B) Currents and histograms under 1Â mM CLT (nÂ =Â 9, pÂ <Â 0.05). (C and D) Currents induced by 1Â mM IMI and the corresponding histograms (nÂ =Â 19, pÂ <Â 0.05). (E and F) Currents and histograms representing currents elicited by 1Â mM TMX (nÂ =Â 14, pÂ <Â 0.05). (G and H) Currents elicited by 1Â mM ACE and Histograms illustrating the increase under bath application of PNU-120596 (nÂ =Â 20, pÂ <Â 0.05). In all case, histograms are meanÂ Â±Â S.E.M. All compounds were applied during 20Â s (black bars). Ctl: represents application of each compound without 1Â Î¼M PNU in the bath.

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