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神经科学

突触膜的微移植以重新激活人突触受体以进行功能研究

Published: July 20, 2022
doi:
10.3791/64024
, , ,
1The Mitchell Center for Neurodegenerative Diseases, Department of Neurology,University of Texas Medical Branch

Summary

该方案表明，通过将突触膜微移植到 非洲爪蟾 卵母细胞中，可以记录α-氨基-3-羟基-5-甲基-4-异噁唑丙酸和γ-氨基丁酸受体的一致和可靠的反应。

Abstract

兴奋性和抑制性离子型受体是离子通量的主要门，它们决定生理神经元通讯过程中突触的活性。因此，已经观察到它们的丰度，功能和与其他突触元素的关系的变化是神经退行性疾病和精神障碍中大脑功能改变和认知障碍的主要相关因素。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，重要的是要记录在患病人脑中保持功能的神经递质受体的电活性。到目前为止，这是评估受体功能病理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，其包括通过注射和后融合到 非洲爪蟾 卵母细胞的膜中，从含有人类受体的速冻人脑组织中重新激活突触膜。该方案还提供了获得α-氨基-3-羟基-5-甲基-4-异噁唑丙酸（AMPA）和γ-氨基丁酸（GABA）受体的一致和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新型详细方法。

Introduction

神经退行性疾病影响很大一部分人群。虽然它们的毁灭性后果是众所周知的，但神经递质受体的功能改变与它们的症状之间的联系仍然知之甚少，这对大脑功能至关重要。个体间变异性，疾病的慢性性质和症状的隐匿发作只是延迟理解许多脑部疾病的一些原因，在这些疾病中，化学失衡有充分的记录1，2。动物模型产生了宝贵的信息，并扩展了我们对进化保守系统中生理学和病理生理学机制的了解;然而，啮齿动物和人类之间的几个种间差异排除了从动物模型到人脑的受体功能的直接外推3。因此，研究天然人类受体的最初努力是由Ricardo Miledi的实验室使用手术去除的组织和冷冻样品开发的。这些初始实验使用包括神经元突触和额外突触受体以及非神经元神经递质受体在内的全膜，尽管它们提供了有关患病状态的重要信息，但人们担心受体的混合会使数据的解释复杂化4，5，6，7。重要的是，突触是许多神经退行性疾病的主要靶点8，9;因此，测试受影响突触的功能特性的测定是获取有关影响突触通讯的疾病相关变化的信息的基础。这里，描述了原始方法的修改：突触膜微移植（MSM），其重点是富集突触蛋白制剂的生理表征，并已成功应用于研究大鼠和人突触体10，11，12，13，14，15.通过这种方法，可以移植曾经在人脑中起作用的突触受体，嵌入到它们自己的天然脂质中，并与它们自己的相关蛋白质队列一起移植。而且，由于MSM数据是定量的，因此可以使用该数据与大型蛋白质组学或测序数据集10集成。

重要的是要注意，突触受体的许多药理学和生物物理分析都是在重组蛋白16，17上进行的。虽然这种方法可以更好地了解受体的结构 – 功能关系，但它无法提供有关神经元中发现的复杂多聚体受体复合物及其疾病变化的信息。因此，天然蛋白和重组蛋白的组合应该提供更全面的突触受体分析。

有许多方法可以制备突触体10，11，12，13，14，15 ，可以根据实验室的要求进行调整。该方案首先假设突触体富集制剂被分离并准备用于微移植实验。在实验室中，按照制造商的说明使用 Syn-Per 方法。这是因为电生理学实验中的高再现性10，11。还有大量文献解释如何分离 非洲爪蟾 卵母细胞18，19，其也可以购买准备注射20。

Protocol

所有研究均按照机构指南进行，并得到加州大学欧文分校动物护理和使用委员会（IACUC-1998-1388）和德克萨斯大学医学分会（IACUC-1803024）的批准。加州大学欧文分校阿尔茨海默病研究中心（UCI-ADRC）提供了来自非阿尔茨海默病（AD）大脑（女性，74岁，死后间隔2.8小时）和AD脑（女性，74岁，死后间隔4.5小时）的颞叶皮层。UCI-ADRC获得了大脑捐赠的知情同意。 注意：未固定的人脑?…

Representative Results

在注射后几个小时内，携带其神经递质受体和离子通道的突触膜开始与卵母细胞质膜融合。图1显示了将AMPA和GABAA受体微移植到非洲爪蟾卵母细胞中的记录。对于大多数分析，使用来自不同青蛙的两到三批卵母细胞来测量每个样品中两个或三个卵母细胞的反应，每个样品总共六到九个卵母细胞。这是为一大群人类受试者观察群体差异而完成的。分析很简单，可?…

Discussion

需要分析来自人类大脑的天然蛋白质复合物，以了解脑部疾病的稳态和病理过程，并制定预防或治疗疾病的治疗策略。因此，含有快速冷冻样本的脑库是大量且大部分未开发的丰富生理信息29，30的宝贵来源。使用死后组织的最初担忧是mRNA或蛋白质降解的明显可能性，这可能会混淆数据的解释。例如，大脑的pH值始终与通过定量实时荧光定量PCR定量mRNA呈…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了NIA / NIH拨款R01AG070255和R01AG073133对AL的支持。我们还感谢加州大学欧文分校阿尔茨海默病研究中心（UCI-ADRC）提供本手稿中显示的人体组织。UCI-ADRC由NIH / NIA拨款P30 AG066519资助。

Materials

For Microinjection
3.5" Glass Capillaries Drummond 3-000-203-G/X
24 well, flat bottom Tissue Culture Plate Thermofisher FB012929
Flaming/Brown type micropipette puller Sutter P-1000
Injection Dish Thermofisher 08-772B
Microcentrifuge Tubes Thermofisher 02-682-002
Mineral Oil Thermofisher O121-1
Nanoject II Drummond 3-000-204
Nylon mesh Industrial Netting WN0800
Parafilm Thermofisher S37440
Stereoscope Fisher Scientific 03-000-037
Syringe Thermofisher 14-841-31
Ultrasonic cleaning bath Thermofisher FS20D
Xenopus laevis frogs Xenopus 1 4217
For Two Electrode Voltage clamp
15 cm long fire polished borosilicate glass capillaries Sutter B200-116-15
Any PC computer or laptop
Low-pass Bessel Filter Warner Instruments LPF-8
Stereoscope Fisher Scientific 03-000-037
Two electrode voltage clamp workstation Warner Instruments TEV-700
ValveLink 8.2 Perfusion Controller Automate Scientific SKU:01-18
WInEDR Free software University of Strathclyde Glasgow https://spider.science.strath.ac.uk/sipbs/software_ses.htm
X Series Multifunction DAQ National Instruments NI USB-6341
Reagents
Calcium dichloride Thermofisher C79
Calcium nitrate tetrahydrate Thermofisher C109
Collagenase Sigma-Aldrich C0130
GABA Sigma-Aldrich A2129
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) Thermofisher BP310
Kainic acid Tocris 0222
Magnesium sulfate heptahydrate Thermofisher M63
Potassium chloride Thermofisher P217
Sodium bicarbonate Thermofisher S233
Sodium chloride Thermofisher S271-1
Ultrafree-0.1 µm MC filter, Amicon
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Tags

Keywords: MicrotransplantationSynaptic MembranesSynaptic ReceptorsFunctional StudiesNeurodegenerative DisordersMental Health DisordersSynaptic CommunicationSynaptic ReceptorsBrain ReceptorsReceptor FunctionalityReceptor ChangesReceptor ActivityInjectionNanoinjectorOocytesSample PreparationSonicationThermoplasticManipulatorFoot PedalPetri DishControl Group
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Miller, B., Powell, A., Gutierrez, B. A., Limon, A. Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies. J. Vis. Exp. (185), e64024, doi:10.3791/64024 (2022).
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