The Mitchell Center for Neurodegenerative Diseases, Department of Neurology, University of Texas Medical Branch.
The Mitchell Center for Neurodegenerative Diseases, Department of Neurology, University of Texas Medical Branch;
J Vis Exp. 2022 Jul 20(185). doi: 10.3791/64024.
Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and γ-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.
离子型受体包括兴奋性和抑制性受体，它们是决定生理神经元通讯中突触活动的离子流的主要门控。因此，在神经退行性疾病和精神障碍中，观察到这些受体的丰度、功能及其与其他突触成分的关系的改变，与大脑功能的改变和认知障碍密切相关。了解兴奋性和抑制性突触受体的功能如何被疾病改变对于开发有效的治疗方法至关重要。为了获得与疾病相关的信息，记录在患病人类大脑中仍然具有功能的神经递质受体的电活动非常重要。到目前为止，这是评估受体功能病理性改变的最接近的方法。在这项工作中，提出了一种进行突触膜微移植的方法，该方法包括将含有人类受体的冷冻人脑组织中的突触膜重新激活，通过注射并随后融合到非洲爪蟾卵母细胞膜中。该方案还提供了获得 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）和 γ-氨基丁酸（GABA）受体一致且可靠反应的方法学策略，以及用于归一化和严格数据分析的详细新方法。
