高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价
1Molecular Neuropharmacology and Genetics Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences,University of Copenhagen, 2Laboratory of Neuropsychiatry, Psychiatric Center Copenhagen and Department of Neuroscience and Pharmacology,University of Copenhagen
Summary
触多巴胺摄取和高效液相色谱分析是通过评估多巴胺转运体的功能和纹组织中多巴胺的水平来研究小鼠体内多巴胺稳态的实验工具,分别.在这里, 我们提出了测量多巴胺组织含量和评估多巴胺转运体功能的协议。
Abstract
多巴胺 (DA) 是一种调节神经递质控制运动活动, 奖励过程和认知功能。多巴胺能 (DAergic) 神经与一些中枢神经系统相关疾病如帕金森氏症、注意力缺陷多动障碍和药物成瘾有密切关系1,2 ,3,4。划定疾病机制涉及 da 失衡是严重依赖于动物模型, 以模拟疾病的方面, 因此, 评估 da 稳态的特定部分的协议是重要的提供新的见解和可能的治疗这些疾病的目标。
在这里, 我们提出两个有用的实验协议, 当结合提供一个功能出的 DAergic 系统的小鼠。通过 da 水平和多巴胺转运器 (DAT) 功能的评估, 获得了 da 稳态的生物化学和功能参数5。在调查 da 系统时, 从成人大脑中可靠地测量 da 的内在水平的能力是必不可少的。因此, 我们提出如何对小鼠脑组织进行高效液相色谱 (HPLC) 测定 DA 的含量。我们执行的实验, 从背纹状体 (dStr) 和伏隔核 (NAc), 但该方法也适用于其他 DA 支配脑区。
DAT 是再进入前终端的关键, 从而控制了 da 的时间和空间活动。在评估 da 稳态时, 了解纹状体中的 DAT 的水平和功能是很重要的。在这里, 我们提供了一个协议, 允许使用触的6 DA 吸收法同时推断表面层和功能的信息。
目前的方法结合标准的免疫协议, 为研究人员提供了相关的 DAergic 系统的描述工具。
Introduction
多巴胺 (DA) 是一种调节神经递质关键的运动行为, 奖励和认知功能1,7,8,9。DA 稳态失衡与一些神经精神疾病有牵连, 如注意缺陷多动障碍、药物成瘾、抑郁症和帕金森氏病1。DA 是释放从前神经元进入突触裂, 它绑定到和激活受体的前突触后膜, 从而进一步传递信号。在释放后突触中 DA 的水平由 DAT3,10进行空间和世俗控制。运输者固 da 从细胞外空间, 从而维持生理 da 水平3,11。在小鼠中, hyperdopaminergic 的基因去除导致突触 da 水平升高, 细胞内 da 池衰竭, 突触后 DAergic 信号发生深刻变化,10,12。
这里, 提出了两个单独的协议, 一个测量 DA 组织含量的方法, 另一个是评估 DAT 的功能. 结合加百利 et al. 的表面法测定法13这两种方法提供了有关 da内容和功能级别的 DAT, 以彻底评估 DA 稳态。利用这些方法可以对各种转基因小鼠或疾病模型的 DA 稳态进行定性和描述。这些工具已被实施和优化, 并在我们的实验室标准使用。目前的化验结果, 以调查的影响 da 稳态改变的 C 终端的 DAT14或表达的重组在酪氨酸羟化酶 (TH) 启动子5。
Protocol
丹麦动物实验视察员的指导方针 (许可号: 2017-15-0201-01160) 被遵循, 并在当地动物福利监督下在一个完全 AAALAC 的认证设施中进行实验委员会. 1. 触多巴胺摄取量 (方法 1) 注意: 此协议用于两个大脑的并行评估, 但可以成功地用于执行触 DA 摄取实验与四大脑并行. 准备 标签 48 1.5 毫升微型离心机管每 表 1 (每个?…
Representative Results
当前 DA 摄取协议 (图 1) 包括评估突中的 DAT 功能所需的所有步骤。我们的 DA 吸收方法 (图 2) 的代表性数据描述了带有未调整数据的饱和曲线 (图 2B) 和调节的数据 (图 2A)。饱和曲线显示野生型小鼠的摄取量。通常一个会使 DA 吸收与突变的小鼠相比, 这将导致一个饱和曲线为每个基因?…
Discussion
这篇手稿描述了有用的实验协议, 以描绘 DA 稳态在任何鼠标模型的选择。我们提供了详细的协议, 以测量 da 的脑组织从小鼠使用 HPLC 和触 da 吸收评估功能 da 运输通过 DAT。下面将阐述 HPLC 实验和触 DA 吸收法的程序、协议和限制。
触吸收协议可以为 dat 的功能提供有用的洞察力. 结合表面法实验13, 可以获得关于 dat 的总数量、表面级别和功能的知识。考虑到 da…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 UCPH 2016 方案的支持 (U.G., ar, K.J.), Lundbeck 基金会 (大副) Lundbeck 生物膜基金会中心纳米 (U.G.), 国家卫生研究院赠款 P01 DA 12408 (U.G.), 丹麦独立研究委员会-医学科学 (U.G.)。
Materials
| COMT inhibitor | Sigma Aldrich, Germany | RO-41-0960 | For synaptosomal DA uptake protocol |
| [3H]-Dopamine | Perkin-Elmer Life Sciences, Boston, MA, USA | NET67-3001MC | For synaptosomal DA uptake protocol |
| Glass microfiber filters | GF/C Whatman, GE Healthcare Life Sciences, Buckinghamshire | 1822-024 | For synaptosomal DA uptake protocol |
| HiSafe Scintillation fluid | Perkin Elmer | 1200-437 | For synaptosomal DA uptake protocol |
| MicroBeta2 | Perkin Elmer | For synaptosomal DA uptake protocol | |
| BCA Protein Assay kit | Thermo Scientific Pierce | 23225 | For synaptosomal DA uptake protocol |
| HEPES | Sigma Life Science | H3375 | For synaptosomal DA uptake protocol |
| Sucrose | Sigma Life Science | S7903 | For synaptosomal DA uptake protocol |
| NaCl | Sigma Life Science | S3014 | For synaptosomal DA uptake protocol |
| KCl | Sigma Life Science | P9541 | For synaptosomal DA uptake protocol |
| CaCl2 | Merck KGaA | 10043-52-4 | For synaptosomal DA uptake protocol |
| MgSO4 | Sigma Life Science | 63065 | For synaptosomal DA uptake protocol |
| Ascorbic Acid | Sigma Life Science | A0278 | For synaptosomal DA uptake protocol |
| D-Glucose | Sigma Life Science | G7021 | For synaptosomal DA uptake protocol |
| Pargyline | Sigma Aldrich | P-8013 | For synaptosomal DA uptake protocol |
| Desipramine | Sigma Aldrich | D3900 | For synaptosomal DA uptake protocol |
| Dopamine | Sigma Life Science | H8502 | For synaptosomal DA uptake protocol |
| Cocaine | Sigma Life Science | C5776 | For synaptosomal DA uptake protocol |
| Brain matrix | ASI instruments | RBM2000C | For synaptosomal DA uptake protocol |
| Cafano mechanical teflon disrupter | Buch & Holm | Discontinued | For synaptosomal DA uptake protocol (homogenization) |
| Antec Decade (Amperometric detector) | Antec, Leiden, The Netherlands | Discontinued: new model DECADE Elite / Lite™ Electrochemical Detector type 175 and 176 | For HPLC protocol |
| Avantec 0.22 μm glass filter | Frisenette ApS, Denmark | 13CP020AS | For HPLC protocol |
| Column: Prodigy 3 μ ODS-3 C18 | Phenomenex, YMC Europe, Chermbeck, Germany | Part Number:00A-3300-E0 | For HPLC protocol |
| LC solution software | Shimadzu | LabSolutions Series Workstation | For HPLC protocol |
| Perchlor acid 0.1M | Fluka Analytical | 35418-500ml | For HPLC protocol (Tissue preparation) |
| EDTA | Sigma | E5134-50g | For HPLC protocol |
| Natriumdihydrogenphosphar | Bie&Berntsen | 1.06346 1000g | For HPLC protocol |
| Sodium 1-octanesulfonate monohydrate | Aldrich | 74885 -10g | For HPLC protocol |
| Acetonitrile, isocratic HPLC grade | Scharlau | AC03402500 | For HPLC protocol |
| Filtre 0.22um | Frisenette ApS, Denmark | Avantec 13CP020AS | For HPLC protocol (Tissue preparation) |
| ortho-Phosphoric acid 85% | Merck | 1.00563. 1000ml | For HPLC protocol |
| Electrode | Antec, Leiden, The Netherlands | AN1161300 | For HPLC protocol (see manual online) |
| Detector program on DECADE II electrochemical detector | Antec, Leiden, The Netherlands | Lite™ Electrochemical Detector type 175 and 176 | For HPLC protocol |
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Cite This Article
Jensen, K. L., Runegaard, A. H., Weikop, P., Gether, U., Rickhag, M. Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake. J. Vis. Exp. (127), e56093, doi:10.3791/56093 (2017).