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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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신경과학

一种基于板的测定法，用于测量急性脑切片中内源性单胺释放

Published: August 11, 2021
doi:
10.3791/62127
, , ,
1Department of Medicine, School of Medicine,Universidad de Atacama, 2Department of Molecular, Cellular, and Biomedical Sciences,City University of New York School of Medicine at City College, 3Department of Pharmacology and Therapeutics,University of Florida College of Medicine

Summary

该方法引入了一种使用急性脑切片检测内源性单胺释放的简单技术。该装置使用含有组织支架的48孔板进行单胺释放。释放的单胺通过HPLC结合电化学检测进行分析。此外，该技术还为药物发现提供了一种筛选方法。

Abstract

单胺类神经递质与许多神经系统和精神疾病有关。这些条件的动物模型已经显示出单胺神经递质释放和摄取动力学的改变。技术上复杂的方法，如电生理学，快速扫描循环伏安法（FSCV），成像，体内微透析，光遗传学或使用放射性是研究单胺功能所必需的。这里介绍的方法是一种优化的两步法，用于检测急性脑切片中的单胺释放，使用含有组织支架的48孔板来检测单胺释放，并使用高效液相色谱联用电化学检测（HPLC-ECD）进行单胺释放测量。简而言之，使用组织切片器或振动器获得包含感兴趣区域的大鼠脑切片，包括前额叶皮层，海马体和背纹状体。从整个大脑中解剖这些感兴趣的区域，并在含氧的生理缓冲液中孵育。在整个实验过程中，通过3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物（MTT）测定法检查了活力。急性解剖的大脑区域在不同的药物条件下孵育，已知这些药物条件通过转运蛋白（苯丙胺）或通过活化胞细胞囊泡释放（KCl）诱导单胺释放。孵育后，收集上清液中释放的产物并通过HPLC-ECD系统进行分析。在这里，通过HPLC从急性脑切片中检测到基础单胺释放。该数据支持先前的体内和体外结果，表明AMPH和KCl诱导单胺释放。该方法对于研究与单胺转运蛋白依赖性释放相关的机制特别有用，并且提供了以快速和低成本的方式筛选影响单胺释放的化合物的机会。

Introduction

过多的神经系统和精神疾病与单胺类神经递质（多巴胺[DA]，5-羟色胺[5-HT]，去甲肾上腺素[NE]）稳态的失调或维持不足有关1，2，3。这些疾病包括但不限于抑郁症1，2，精神分裂症2，焦虑症2，成瘾4，更年期5，6，7，疼痛8和帕金森病3。例如，几种更年期大鼠模型表明，海马体、前额叶皮层和纹状体内单胺失调或减少可能与抑郁和认知能力下降有关，这在经历更年期的女性中可见。这些模型中单胺的失调已经使用HPLC-ECD进行了广泛的检查，尽管这些研究没有区分测量的神经递质含量与神经递质释放5，6，7。单胺通过Ca2 +依赖性囊泡释放9经典地释放到细胞外空间，并通过其各自的质膜再摄取系统（多巴胺转运蛋白，DAT;血清素转运蛋白，SERT;去甲肾上腺素转运蛋白，NET）10，11循环。相反，数据表明，这些转运蛋白能够释放或排出单胺，因为已知苯丙胺（AMPH）和3，4-亚甲二氧基甲基苯丙胺（MDMA）等滥用药物分别通过其转运蛋白系统释放DA和5-HT12，13，14，15，16，17.因此，对单胺释放动力学的正确机制理解对于开发特定和靶向的药物治疗至关重要。

已经采用了广泛的技术来研究单胺释放，例如快速扫描循环伏安法（FSCV）18，体内微透析13，成像19，放射性标记的单胺预孵育20，光遗传学，以及最近的遗传编码荧光传感器和光度测定法21，22.FSCV和体内微透析是用于研究单胺释放的主要技术。FSCV用于研究刺激的DA在急性脑切片和体内的胞吐释放23。由于FSCV使用电极来刺激或唤起释放，因此神经递质释放的主要来源是Ca2 +依赖性囊泡释放18，24，25，26，27，28，29，30，31.体内微透析与HPLC相结合，使用放置在感兴趣大脑区域的探针测量细胞外神经递质水平的变化13，32。与FSCV类似，体内微透析的一个主要限制是难以确定神经递质释放的来源：Ca2 +依赖性囊泡释放或转运蛋白依赖性。值得注意的是，这两种方法都可以直接测量单胺释放。通过光遗传学的最新进展，研究表明，在短时间内检测到5-HT和DA释放，具有精致的细胞类型特异性21，22。然而，这些策略需要复杂而昂贵的技术和设备，并间接测量单胺释放，特别是通过单胺与受体结合。此外，放射性标记的单胺也用于研究单胺动力学。放射性标记的单胺可以预加载到各种模型系统中，例如异源细胞过度表达每个单胺转运蛋白20，33，34，35，36，37，38，39，40，原代神经元20，突触体33，39，41，42，和急性脑切片43，44。然而，放射性对实验者造成潜在的伤害，并且氚标记的分析物可能无法忠实地概括内源性单胺动力学45，46。超聚变系统与HPLC-ECD等离线检测方法相结合，允许从多个组织来源检测单胺。在这里，该协议提供了一种优化和低成本，简单和精确的方法，使用急性脑切片直接测量内源性基底和刺激的单胺释放。

急性脑切片允许测试机械假设，主要是因为它们保留了体内解剖学微环境，并保持完整的突触47，48，49，50，51，52。在一些研究中，急性脑切片或切碎的脑组织已与使用KCl刺激Ca2 +介导的释放的超融合技术结合使用53，54，55，56。超聚变系统对于推进该领域对神经递质释放机制（包括单胺）的理解至关重要。然而，这些系统相对昂贵，可用于组织分析的腔室数量从4-12不等。相比之下，这里介绍的方法价格低廉，可以测量48个组织样品，并且可以改进以使用多达96个组织样品。48孔板内的每个孔都包含组织支架，这些组织支架使用过滤器将释放的产物从组织中分离出来，然后通过HPLC-ECD收集和分析释放的单胺。重要的是，该方法允许在用调节单胺释放的药理剂治疗后，同时测量来自不同大脑区域（如前额叶皮层，海马体和背纹状体）的5-HT，DA和NE释放。因此，实验者可以使用廉价的多孔系统回答多个问题，该系统增加了测试样品的数量，从而减少了使用的动物数量。

Protocol

所有实验，包括动物处理和组织收集，都是根据佛罗里达大学和纽约城市学院机构动物护理和使用委员会（IACUC）按照批准的协议201508873（UF）和1071（CCNY）进行的。有关试剂和缓冲液，请参阅 补充文件。 1. 准备急性大鼠脑切片 注意：在该实验中，使用成年雄性大鼠（250-350g）。然而，这种设置对于不同的发育点，雌性大鼠和其他物种是有效的…

Representative Results

该技术描述了使用脑切片来测量内源性单胺的释放，使用HPLC和基于具有内部组织支架的48孔板的电化学检测。实验设置如图 1 和 图2所示。最初，为了确保在实验结束时的组织活力，进行了MTT（3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物，四氮唑）测定。经过功能实验后，与与1%Triton X-100（细胞死亡条件）孵育的急性脑切…

Discussion

多年来，单胺释放测量已经在许多系统中进行，例如异源细胞，神经元培养物，脑突触体，离体急性脑切片和整个动物13，20，41，42，58，64，65，66，67，<sup class…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了基金会发起基金N 11191049 J.A.P.和NIH赠款DA038598对G.E.T.的支持。

Materials

48 Well plate NA NA Assay
Acetonitrile Fischer Scientific A998-1 Mobile Phase
Calcium Chloride Ahydrous Sigma Aldrich C1016 Modified Artifical Cerebrospinal Fluid OR Efflux Buffer
Clarity Software Anetc
Citric Acid Sigma Aldrich Mobile Phase
D-(+)-Glucose Sigma 1002608421 Dissection Buffer
DMF Sigma Aldrich D4551 MTT Assay
EDTA-Na2 Sigma Aldrich Mobile Phase
GraphPad Software Graphpad Software, Inc Statistical Analysis
Glycerol Sigma Aldrich G5516 Lysis buffer
HEPES Sigma Aldrich H3375 Lysis buffer
HPLC, Decade Amperometric Anetc HPLC, LC-EC system
HPLC Amuza HPLC HTEC-510.
L-Asrobic Acid Sigma Aldrich A5960 Dissection Buffer
Magnesium Sulfate Sigma 7487-88-9 KH Buffer
Microcentrifuge Filter Units UltraFree Millipore C7554 Assay – 6 to fit in 48 well plate
MTT Thermo Fisher M6494 MTT Assay
Nanosep VWR 29300-606 Assay; protein assay
Octanesulfonic acid Sigma Aldrich V800010 Mobile Phase
Pargyline Clorohydrate Sigma Aldrich P8013 Modified Artifical Cerebrospinal Fluid OR Efflux Buffer
Phosphoric Acid Sigma Aldrich Mobile Phase
Potassium Chloride Sigma 12636 KH Buffer
Potassium Phosphate Monobasic Sigma 1001655559 KH Buffer
Precisonary VF-21-0Z Precissonary Compresstome
Protease Inhibitor Cocktail Sigma Aldrich P2714 Lysis buffer.
Sodium Bicarbonate Sigma S5761 Dissection Buffer
Sodium Bicarbonate Sigma Aldrich S5761 Dissection Buffer
Sodium Chloride Sigma S3014 KH Buffer
Sodium Dodecyl Sulfate Sigma Aldrich L3771 Lysis buffer
Triton X-100 Sigma Aldrich T8787 MTT Assay / Lysis buffer
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Endogenous Monoamine ReleaseBrain SlicesPlate-based AssayDorsal StriatumHippocampusEfflux BufferPharmacological ConditionsVesicular ReleaseTransporter-mediated Release
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Pino, J. A., Awadallah, N., Norris, A. M., Torres, G. E. A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices. J. Vis. Exp. (174), e62127, doi:10.3791/62127 (2021).
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