切片热门：成人急性脑切片在生理温度
Summary
In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.
Abstract
在这里，我们提出了一个协议，准备对急性脑片。这个程序是用于电生理膜片钳实验的关键元件，其在很大程度上决定结果的质量。它已经显示，省略了切割过程中的冷却步骤是有利的，获得健康的切片和细胞，特别是当与从成熟动物的高度髓脑结构处理。虽然确切的机制，使高温下支持神经健康时只能推测，它按理说，只要有可能，在切片时的温度应接近生理条件，以防止温度有关的文物。该方法的另一个重要的优点是该过程的简单性，因此，制备时间短。在证明方法成年小鼠进行使用，但相同的过程可以与年轻小鼠以及大鼠施用。此外，下面的补丁CL安培实验是在水平的小脑切片进行的，但同样的过程也可以用在其它的面以及大脑的其他后部区域内使用。
Introduction
该方法的目的是为了得到高质量的急性脑切片的体外电生理实验，尤其是当使用成人或甚至老的动物。
急性脑切片的方法，因为两个优雅的句子描述Skrede和Westgaard 1，已成为现代神经科学研究的基础之一，并在全球范围内采用无数的变化。切片的质量反映在活每片神经元的数目，该时间周期，在此期间细胞保持它们的电生理学和形态学特性，以及在组织中的完整性。此外，最大持续时间为稳定记录依赖于切片的质量。因此，沿几十年来，原来切断方法有了进一步的发展由个体研究团体切割2-10，常常通过切割的组合物的复杂的修改后，提升片恢复öR恢复解决方案（例如，添加抗坏血酸，硫脲或甚至H 2 O 2），以及心内预灌注动物用冷却的生理溶液。
正如最近的研究显示11，切片过程中的生理温度似乎比冷却到神经元的健康更有益;与成年（2-8月）的啮齿动物工作时的改善是最引人注目的。避免剧烈的温度变化时防止工件因温度依赖性过程中的细胞，如可塑性13和离子通道动力学13,14。这些变化可能会影响膜电位和细胞内钙信号，穗门槛，穗形。
“热”急性切片制备这里介绍的方法是用于获得高品质的急性脑切片从任何脑区域，包括小脑，皮层，海马，脑干晶核16的一般步骤</ SUP>以及嗅球，无论是在大鼠和小鼠。
值得注意的是，在生理温度下的切片过程要求的切削刀片的振动几乎完全水平，是没有任何结构上的缺陷。这样的精确度可能无法实现与老款切片;在这种情况下，我们建议在进行切片准备在冷冻 – 寒冷的条件下作为低温度似乎使机械损伤，在代谢异常的费用的组织更耐即使。
Protocol
Representative Results
Discussion
我们证明了在生理，而不是冰冷的温度筹备急性脑切片的小鼠的方法。
已经显示11，当与制备的那些与冷的条件下相比在温暖条件下获得的切片的质量是优异的，其前提是该切片机叶片具有最小的垂直振动。切片在生理温度可以防止生理伪影引起的低温，例如那些涉及到改变在代谢过程17-19引起可在单细胞的像差以及网络行为。此外，即使在切片过程显然应避…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.
Materials
| Name | Company | Catalog # | Comments |
| Pentobarbital | CTS | 170066 | Concentration: 60 mg / ml in physiological saline. |
| Big scissors | FST | 14001-16 | Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation |
| Iris scissors | Prestige medical | 48,148 | Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 – 2 cm |
| Fine tip forceps | FST | 11254-20 | |
| Scalpel | FST | 91003-12 | |
| Scalpel blade #11 | FST | 10011-00 | |
| Small spatula | Fisher | 2350 | |
| Filter paper | Any laboratory brand can be used. | ||
| Petri dishes | Duroplan | Z231509-1 | |
| Glass beakers | SCHOT | 10022846 | |
| Pasteur pipette | Maple Leaf Brand | 14672-029 | |
| Super glue | LOCTITE | 4091361/1 | |
| Slicer | Campden | 7000-smz | |
| Ceramic slicing blade | Campden | 7550-1-C | |
| Magnetic heater/stirrer | For heating up the SPS for the procedure | ||
| Electric kettle | For heating up water for temperature control | ||
| Slice recovery chamber + heating unit | Warner instruments | BSC-HT + BSC-BUW | Home-built models may also be used. |
| Thermometer | For monitoring SPS temperature during dissection and slicing |
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