小鼠大脑中耳蜗的定位
Summary
胁迫位点是参与各种生理过程的一个小的神经元簇。在这里, 我们描述了一个方案, 准备小鼠大脑部分, 用于研究蛋白质和金属在这个细胞核。
Abstract
逼位点 (lc) 是去甲肾上腺素产生神经元的主要枢纽, 调节许多生理功能。lc 的结构或功能异常会影响几个大脑区域, 包括皮层、海马和小脑, 并可能导致抑郁、双相情感障碍、焦虑以及帕金森病和阿尔茨海默病。这些疾病通常与金属失平衡有关, 但金属在 lc 中的作用只被部分理解。需要对 lc 进行形态和功能研究, 以更好地了解人体病理和金属的贡献。小鼠是一种广泛使用的实验模型, 但小鼠 lc 体积小 (直径约 0.3 mm), 对于非专家来说很难识别。在这里, 我们描述了一个逐步免疫组织化学为基础的协议, 以本地化在小鼠大脑中的 lc。多巴胺-β-羟化酶 (dbh), 或者酪氨酸羟化酶 (th), 这两种酶在 lc 中高度表达, 被用作大脑切片的免疫组织化学标志物。与含 lc 切片相邻的部分可用于进一步分析, 包括用于形态学研究的组织学、代谢检测以及 x 射线荧光显微镜 (xfm) 的金属成像。
Introduction
费鲁厄斯 (lc) 位点 (lc) 是脑干的一个重要区域, 也是去甲肾上腺素 (ne) 产生的主要部位1。lc 将整个大脑2的投影发送到皮层、海马体和小脑3 , 并调节主要的生理过程, 包括生理节律4,5, 注意力和记忆6,压力7, 认知过程8, 和情绪9,10。lc 功能障碍与神经和神经精神障碍有 11例, 包括帕金森病 12、13、14、阿尔茨海默病14、抑郁症15 16,17, 双相情感障碍18,19,焦虑20,21, 22,23,24. 鉴于这些作用, 对 lc 的分析对于研究其功能和功能障碍至关重要。
小鼠被广泛用于生理和病理生理过程的研究。由于体积小, 小鼠 lc 的平均直径约为 300μm, 因此难以定位结构。在脑切片过程中, lc 很容易在冠状或矢状切片中丢失。现有的研究描述了 lc 在动物中的识别没有提供一个分步的协议, 一个非专家可以遵循1,25。因此, 为了提供 lc 本地化的指导, 我们描述了一个协议, 我们开发的协议在多个应用程序的鼠标大脑中定位此区域 (图 1, 图 2, 图 3)。该方案应用精心控制的脑切片和免疫组织化学检测 dbh26,27, 或交替 th24, 这两种酶在 lc28中高度富集。一旦 lc 通过免疫组织化学定位, 相邻的大脑切片可用于进一步的研究, 包括形态学和代谢分析, 以及通过 x 射线荧光显微镜 (xfm)29进行金属成像研究。我们将 xfm 描述为本协议中的一个示例 (图 3)。
Protocol
约翰·霍普金斯大学动物护理和使用 (acuc) 议定书编号 m017m385 批准了对动物的研究。 1. 脑切片 为了固定, 用3% 异氟醚麻醉小鼠。 用异氟醚滴浸泡棉球, 并将其放入15毫升的微离心管中。将动物的鼻子放入试管中, 让其吸入异氟醚。检查麻醉的深度是否缺乏对脚趾夹紧的反应。 将动物放在背部, 并通过用 t 针固定其四肢, 同时进入其腹部, 使其固定?…
Representative Results
金属稳态的变化 (如铜、铁、锌和锰) 经常在神经紊乱中观察到, 包括 lc34、35的变化。因此, 确定大脑中的金属含量对于了解疾病机制是必要的。使用所述协议生成的大脑部分可用于量化 lc 中的铜和其他金属的水平, 并将其与 lc 以外区域的水平进行比较。(图 3)。在我们的例子中, 测量了通过 lc、磷酸盐、钾?…
Discussion
正确定位样品是本协议中的关键一步。因为我们使用大脑背侧表面的解剖特征来定位 lc (小脑和下大肠杆菌之间的边界), 因此正确对齐各个部分是很重要的。这就需要小心, 把大脑适当地设置在老鼠的大脑切片器矩阵中。我们建议切割 ~ 500μm 以上的组织前和后 lc, 以避免丢失细胞核。最常见的错误是削减太少的部分, 导致完全错过 lc。因此, 对于第一次遵循此协议, 我们建议切割超过必要的部分。仔…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 abigael muchdididsi 维持了老鼠的殖民地。argonne 国家实验室先进光子源的使用得到了美国能源部、科学办公室、基础能源科学办公室的支持, 合同号为: de-ac02-06ch11357。我们感谢 olga antipova 和 stefan vogt 博士在高级光子源的用户支持和帮助。这项工作由国家卫生研究所向 sl 提供的赠款2r01gm101502 资助。
Materials
| Adult mouse brain slicer matrix | Zivic Instruments | BSMAS001-1 | |
| Anti-rabbit secondary antibody, Alexa Fluor 488 (source – donkey) | Thermo Fisher Scientific | A-21206 | |
| Charged glass slides | Genesee | 29-107 | |
| Confocal microscope | Zeiss | LSM 800 | |
| Cryostat | Microm GmbH | HM 505E | |
| Cryostat cutting blades | Thermo Fisher Scientific | MX35 | |
| Scissors Mini, 9.5cm | Antech Diagnostcs | 503241 | |
| DAPI (4',6-diamidino-2-phenylindole) | Sigma-Aldrich | D9542-10MG | |
| Dopamine β-hydroxylase (DBH) antibody – inhouse production (source – rabbit) | B. Eipper | – | |
| Dopamine β-hydroxylase (DBH) antibody – commercially availabe (source – rabbit) | Cell Signaling | 8586 | |
| Falcon tubes, 50ml | USA Scientific | 339652 | |
| Forane (isofluorane) | Baxter | NDC 1019-360-60 | |
| Forceps Micro Adson | Antech Diagnostcs | 501245 | |
| Hardset mounting media | EM sciences | 17984-24 | |
| Microscope | Pascal | LSM 5 | |
| Multi-well plates, 24 wells | Thermo Fisher Scientific | 930186 | |
| Optimal cutting temperature compound (OCT) | VWR/ tissue tech | 102094-106 | |
| Paraformaldehyde (PFA)/ formalin 10% | Fisher Scientific | SF98-4 | |
| Peel-A-Way disposable embedding molds | Polysciences Inc. | 18646A | |
| Pencil brush | |||
| Phosphate buffered saline (PBS) | Life Tech | 14190250 | |
| Razor blades | Amazon | ASIN: B000CMFJZ2 | |
| Spatulas | Antech Diagnostcs | 14374 | |
| T pins | Office Depot | 344615 | |
| The Mouse Brain in Stereotaxic Coordinates, Paxinos and Franklin, 3rd Edition | Amazon | ISBN: 978-0123694607 | |
| Triton-X 100 (to prepare PBSD) | Sigma-Aldrich | T8787 | |
| Tween 20 | Sigma-Aldrich | P7949-500ml | |
| Tyrosine hydroxylase (TH) antibody (source – rabbit) | EMD Millipore | AB152 | |
| Ultralene thin film for XRF | SPEX Sample Prep | 3525 | |
| Wide-field fluorescent microscope | Zeiss | Axio Zoom.V16 | |
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Cite This Article
Schmidt, K., Bari, B., Ralle, M., Washington-Hughes, C., Muchenditsi, A., Maxey, E., Lutsenko, S. Localization of the Locus Coeruleus in the Mouse Brain. J. Vis. Exp. (145), e58652, doi:10.3791/58652 (2019).