禽流感胚胎听神经听觉神经纤维的选择性跟踪
Summary
在这里，我们描述了一个显微切割技术，用荧光染料注射到声早期鸡胚神经节选择性地跟踪听觉神经轴突的神经纤维和后脑。
Abstract
鸡胚是一种广泛使用的外周和中枢的神经节细胞预测模型的研究。在听觉系统，听觉选择性标记的第八次颅神经轴突内将提高研究听觉中枢电路的发展。这种方法是具有挑战性的，因为多种感官的内耳器官的第八次神经。此外，可靠区分的标记，听觉与前庭禽流第八次神经轴突内还没有得到确定。听觉和前庭通路不能区分功能在早期胚胎中，感觉诱发反应不存在之前形成的电路。中心突出的第八次神经轴突已经在一些研究中追踪，伴随着但听觉神经轴突标签的标签从其他的第八次神经组件2,3。在这里，我们描述的方法，顺行追踪声节有选择地拉贝升听觉的第八次神经轴突内。首先，在为期8天的鸡胚沉浸在氧合人工脑脊液的前头部的局部解剖，解剖标志确定的蜗管。接着，罚款拉玻璃微被定位成注入少量罗丹明葡聚糖胺进入管和相邻的位于较深的区域中的声学的神经节细胞。在注射30分钟后，听觉神经轴突集中到后脑被跟踪，以后可以可视化的组织学准备。这种方法提供了一个有用的工具，周边听觉中枢电路形成发育研究。
Protocol
1。准备好以下的解剖工具和试剂人工脑脊液（人工脑脊液; 130 mM氯化钠，3 mM KCl中，1.2毫摩尔KH 2 PO 4，20毫摩尔的NaHCO 3，3 mM的HEPES，10mM的葡萄糖，2毫摩尔的CaCl 2，1.3 mM的用MgSO 4）用95％O连续输注2/5％CO 2中 ，在室温下。输液，补到2/3的500毫升广口的Nalgene瓶的盖子钻了一个洞。坦克通过管道将被附加的玻璃干起泡，贯通通过的jar盖中的孔的?…
Representative Results
第八次神经和神经本身的解剖结构的组成部分是复杂的，错综复杂的（ 图1，图3）。通过选择性地跟踪从声学的神经节细胞所产生的纤维，可以干净地跟踪段的第八次神经，以及初级听觉传入脑干内区别于他们的前庭同行（ 图2，图3）。同样地，这种技术可用于研究声学的神经节细胞的周缘凸部（ 图3G），或修改，以研究从个别前庭器官所产生的凸起。 ，因…
Discussion
第八次神经的早期发展研究已部分限制，因为很难确定胚胎从多个不同的神经轴突产生。一些研究探讨了指导听觉和前庭感觉细胞和神经节细胞的命运在早期发育过程中的分子信号，5,11,12，但还没有得到确定的调节中央神经支配的进程。声神经节细胞预测报告通常描述外围过程到感觉上皮13日至15日 ，而被称为中央工艺主要核突出。中央突出第八细分在胚胎发育过程中的研究也常…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者要感谢坎迪斯谢医生的建议和援助，对鸡内耳解剖成像技术和多丽丝吴医生的专业知识，在早期胚胎发育。这项工作是由美国国家科学基金会，美国国立卫生研究院IOS-0642346 T32-DC010775，NIH，NIH R01-DC010796，T32-GM008620和美国能源部GAANN P200A120165的支持。
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| Polystyrene Weigh Dish | Fisher Scientific | 02-202-101 | |
| Petri Dish, 35 X 10 mm | Fisher Scientific | 50820644 | Use to make silicone dissection dish |
| Sylgard Silicone Elastomer Kit | World Precision Instruments | SYLG184 | Coat Petri to make dissection dish |
| Dissection Pins | Various | Holds embryo in place during dissection | |
| NaCL | Various | part of aCSF recipe | |
| KCl | Various | part of aCSF recipe | |
| KH2PO4 | Various | part of aCSF recipe | |
| NaHCO3 | Various | part of aCSF recipe | |
| Glucose | Various | part of aCSF recipe | |
| CaCl2 | Various | part of aCSF recipe | |
| MgSO4 | Various | part of aCSF recipe | |
| Container for aCSF. Suggest translucent wide-mouth Nalgene jar, 500 ml (16 oz) with lid. | CPLabSafety | QP-PLC-03717 | Drill hole opening in top of lid for glass bubling stem to penetrate liquid |
| Empty 5 ml glass vial or comparable transparent vial | American Pharmaceutical Partners, Inc | 6332300105 | Use during aCSF incubation to keep samples separate from each other and from the bubbling stream |
| Tank of carbogen (95%O2 / 5%CO2) connected by tubing to bubbler | Various | Attach by tubing to glass stem bubbler for infusion into aCSF | |
| Glass stem bubbler | Various | To infuse carbogen into aCSF | |
| Curved-tip forceps | World Precision Instruments | 501008 | To remove embryo head from egg |
| Two fine-tip forceps | World Precision Instruments | 501985 | For micro-dissection |
| 50 ml Beaker | various | ||
| Rhodamine Dextran Amine (RDA) | Invitrogen | various | Fluorescent axon tracer |
| Triton X-100 | ICN Biomedicals | ||
| Phosphate Buffered Saline, (1X PBS) | Various | Standard lab reagent | |
| Thin Wall Glass Capillaries, 1.2 OD, .9 ID 4″ (100 mm) length | World Precision Instruments | TW120F-4 | Load with RDA. Each capillary makes two glass micropipettes |
| Needle / Pipette puller | David Kopf Instruments | Model 720 | Settings used: Heat 16.4, Solenoid 2.2 |
| Picospritzer | Parker Instrumentation | various | Attach by fine tubing to glass micropipette |
| Micromanipulator | Narishige | various | |
| Dissection microscope with fluorescence | Various | ||
| 4% Paraformaldehyde | Various | Standard lab reagent | |
| anti-Neurofilament antibody, optional | Millipore | AB1991 | Follow histological protocol recommended by manufacturer |
| Cryostat and associated materials for sectioning | Leica | various | |
| Epifluorescent microscope for imaging | Zeiss, various |
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Citar este artículo
Allen-Sharpley, M. R., Tjia, M., Cramer, K. S. Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve. J. Vis. Exp. (73), e50305, doi:10.3791/50305 (2013).