鸡脑干切片的制备及文化
Summary
鸡听性脑干是由原子核负责双耳进行无害化处理。一个单一的冠状切片制剂保持整个电路，而培养的方法，在分子，细胞和网络水平研究神经细胞结构和听觉功能的发展提供了一个独特的准备。
Abstract
鸡听性脑干是一个完善的模型系统，已广泛应用于研究谨慎时期的发展1-4以及5-7在中枢神经系统的时空编码机制的解剖和生理听觉处理。
在这里，我们提出了一个方法来准备鸡听性脑干片可用于急性实验程序或长期的实验操作的器官切片文化。
鸡听性脑干是由原子核angularis，magnocellularis，laminaris和优越的橄榄油。这些原子核负责双耳进行无害化处理和单一的冠状片的准备工作，保持整个电路。最终，器官切片文化可以提供机会，如突触活动，前和突触后成分的表达，表达方面控制的兴奋性和差异表达基因操纵一些发展参数
这种方法可用于扩大对神经回路的发展，完善和成熟的一般知识。
Protocol
1。解剖区的制备连续泡沫人工脑脊髓液（学联）的95％O 2 / 5％的CO 2的混合物（pH值在7.2-7.4之间，渗透压295-310 mOsm / L ）。 学联是冒泡，清洁的工作区用70％乙醇。同时清洁vibratome和切片刀片。 （DH 2 O），用蒸馏水冲洗刀片。 清洁液体吸收垫放在解剖与适当的解剖工具面积。 胶琼脂块（琼脂糖40毫克/毫升，即4％，在卫生署2 O）的一个?…
Discussion
几十年来，鸡的脑干急性切片准备工作已用于研究听觉处理9，10 。这种方法提供了大量的体外生理双耳处理的数据，从发展和成熟的状态4，11 ，12。被知道关于这个高度专业化的电路和每个细胞核中的作用时间处理的声音13，14次。事实上，短期的实验操作和特点电路的结构和功能的影响已被证明有利 15 。但是从长远发展的角度，这样的操作不可能和技术?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
现任和前任鲁贝尔的实验室成员。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Chicken ACSF | ||||
| NaCl | Fisher Scientific | M-11624 | 130 mM | |
| NaHCO3 | Fisher Scientific | M-10576 | 26 mM | |
| KCl | Sigma | P-9333 | 3 mM | |
| NaH2PO4 | Sigma | S-8282 | 1.25 mM | |
| Glucose | Sigma | G-7528 | 10 mM | |
| MgCl2 | Fisher Scientific | M33-500 | 1 mM | |
| CaCl2 | Acros Organics | 423525000 | 2 mM | |
| Chicken culture medium (store at 4°C) | ||||
| advanced minimum essential medium (with NEAA, sodium pyruvate at 110 mg/l, without L-glutamate) | Gibco Invitrogen | 12492-013 | 48.00% | |
| Earl’s balanced salt solution | Sigma | E-2888 | 24.00% | |
| L-glutamine (200 mM) | Sigma | G-7513 | 1.00% | |
| Glucose solution (200 g/l, sterile filtered) | Sigma | G-7528 | 2.75% | |
| horse serum (heat inactivated, sterile filtered) | Sigma | H-1138 | 24.00% | |
| Penicillin-streptomycin* | Sigma | P-0781 | 1.00 ml * = Add to 100 ml culture medium if needed to prevent contamination |
|
| Specific equipment | ||||
| Millicell-CM cell culture inserts | PICMORG50 | Millipore | ||
| 6-well plate | 6 Well Cell Culture Cluster | Corning Incorporated | ||
| Incubator | Forma Scientific CO2 Water Jacketed Incubator | Forma Scientific |
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Citazione di questo articolo
Sanchez, J. T., Seidl, A. H., Rubel, E. W., Barria, A. Preparation and Culture of Chicken Auditory Brainstem Slices. J. Vis. Exp. (49), e2527, doi:10.3791/2527 (2011).