Boussaine Khalid, Taha Maria, Nìng Cáinà, Cartereau Alison, Rakotobe Sabine, Mateos-Hernandez Lourdes, Taillebois Emiliane, Šimo Ladislav, Thany Steeve H
University of Orleans, Laboratory Physiology, Ecology and Environment (P2E) USC-INRAE 1328, 1 rue de Chartres, Orléans Cedex, France; ANSES, INRAE, Ecole Nationale Vétérinaire d'Alfort, UMR BIPAR, Laboratoire de Santé Animale, 22 rue Pierre et Marie Curie, Maisons-Alfort, France.
University of Orleans, Laboratory Physiology, Ecology and Environment (P2E) USC-INRAE 1328, 1 rue de Chartres, Orléans Cedex, France.
J Pharmacol Toxicol Methods. 2023 Nov-Dec;124:107473. doi: 10.1016/j.vascn.2023.107473. Epub 2023 Oct 20.
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 ∼ 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 ∼ 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.
硬蜱（蜱科）的中枢神经系统由一个集中融合的神经团组成，称为交感神经节。尽管在过去二十年中，蜱神经生物学的知识有了显著进步，但这是首次对交感神经节中的蜱神经元进行分离和电生理记录。方法：我们开发了一种简单的交感神经节神经元分离方案，并使用全细胞膜片钳来测量乙酰胆碱、尼古丁和毒蕈碱诱导的离子电流。分离出相对较大的神经元（约25μm和约35μm），并使用1mM乙酰胆碱分别诱导出-0.38±0.1nA和-1.04±0.1nA的强内向电流，相应的细胞电容分别约为142pF和188pF。此外，通过约25μm和约35μm的细胞连续施加1mM乙酰胆碱，持续时间增加，导致电流幅度迅速降低。我们还发现，每种神经元类型中，乙酰胆碱诱发的电流都与细胞内钙水平的可逆增加有关。相比之下，1mM毒蕈碱和尼古丁诱导细胞内钙水平强烈且不可逆地增加。本研究为蜱交感神经节神经元的机械分离及其电生理记录提供了概念验证。这种方法将有助于研究蜱神经元的药理学特性，并提供识别药物靶点和有效蜱控制措施所需的工具。
