活的嗅觉途径的功能评价
1Laboratory of Neurobiology, Neuroscience Program, Bellvitge Biomedical Research Institute (IDIBELL),L’Hospitalet de Llobregat, 2Department of Pathology and Experimental Therapy, School of Medicine, University of Barcelona,L’Hospitalet de Llobregat, 3Institute of Neurosciences, University of Barcelona,L’Hospitalet de Llobregat
Summary
为研究神经系统在体内的功能提供了一个独特的平台。我们描述了在正常饲养条件下或受伤后评估生活的幼虫嗅觉信息处理的方法。
Abstract
为研究神经系统的功能提供了一个独特的平台。它们提供了多种实验优势, 例如多种成像方法、电生理技术和行为检测。嗅觉系统特别适合于研究在正常发育过程中建立的突触功能或损伤后的改造功能。在这里, 我们描述的方法来评估处理嗅觉信息的生活幼虫。我们概述了在体内测量的突触前钙反应的结合在分子引导的行为检测的嗅觉球球球肾小球。方法可与嗅觉神经横断相结合, 研究突触连接的重新布线。实验是使用野生类型和转基因动物表达 gfp 记者在中枢神经系统细胞。应用所描述的转基因的方法可以很好地解开定义脊椎动物行为的分子基础。
Introduction
是研究神经系统正常功能的优秀动物模型。透明, 一个完全测序的基因组1,2, 以及可获得的手术, 电生理和成像技术是独特的属性, 希望研究神经元在体内的功能 3.这种动物模型的多种实验可能性通过对感官和运动系统 4,5,6进行的彻底研究说明了这一可能性。一个特别适合的神经元电路, 研究在突触水平的信息处理的许多方面是嗅觉系统 7。首先, 它的突触连接是很好的定义: 嗅觉感受器神经元 (orn) 项目嗅觉灯泡和建立突触接触与树突的二尖瓣簇状细胞在肾小球内产生气味图。其次, 它的 orn 是由一生的神经发生持续产生的, 以保持嗅觉路径8的功能.第三, 由于嗅觉系统显示出巨大的再生能力, 在烧蚀9后能够完全改造它们的嗅球.
本文介绍了将活树中嗅觉肾小球的成像与行为实验相结合的方法, 以研究嗅觉通路的功能。采用本文详细研究了嗅球在嗅觉神经横截面10后肾小球连接的功能恢复问题。在中获得的数据是脊椎动物的代表, 因为嗅觉处理是进化守变的。
所描述的方法是用x. tropicalis进行的, 但它们可以很容易地在x 中实现。莱维斯。尽管成年的较大, 但这两个物种在阶段都非常相似。主要的差异在于基因组水平。月牙的遗传可追踪性较差, 主要由其异倍体基因组和长时间 (约 1年) 决定。相反,热带 x由于生成时间较短 (5-8个月) 和二倍体基因组, 更适合基因改造。介绍了野生动物和三个不同的转基因线的代表性实验: hb9:gfp (x. tropicalis)、Hb9:GFP (x. tropicalis) 和 tub2:gfp (x. laevis)。
目前工作中概述的方法应与xenopus领域的遗传进展一并考虑。所介绍的技术的简单性和易于实现使其特别有用, 用于评估已经描述的突变体11, 以及 crispr-cas9 技术12生成的xenopus线.我们还描述了一种用于横切嗅觉神经的外科手术, 这种手术可以在任何实验室中实施, 可以接触到。用于评估突触前钙反应和嗅觉引导行为的方法需要特定的设备, 尽管以适中的成本提供。方法以简单的形式提出, 以促进其在研究小组中的使用, 并可通过实施改进或与其他技术, 即组织学或遗传方法的联系, 为更复杂的检测奠定基础。
Protocol
所有程序都得到了巴塞罗那大学动物研究伦理委员会的批准。 请注意:按照标准方法13、14饲养热带和小。水是通过在反渗透获得的水中添加商业盐 (见材料表) 来制备的。电导率分别调整为 x .营养素和x. laevis 的 ~ 700μs 和 ~ 1400μs。幼虫可以通过自然交配或体外受精获?…
Representative Results
本文提出了两种互补方法的组合, 对的功能进行体内研究: i) 一种成像突触前钙 2+生命肾小球变化的方法使用荧光钙指示剂的, 和 ii) 气味引导行为检测, 可用于研究对特定水性气味的反应。由于这些方法已被用来评估受伤 10后嗅觉处理的恢复情况, 因此还介绍了一种简单的造嗅觉神经横切方法。 <p class="jove_content" fo:keep-together.within-…
Discussion
本文介绍了一些技术, 这些技术对于研究活中嗅觉通路的功能很有用。目前的议定书对那些工作或能够进入xenopus的实验室特别有用;然而, 这也是有趣的那些研究神经元再生和修复的细胞和分子基础的研究人员。在中获得的结果可以与其他脊椎动物模型中收集的数据结合起来, 以确定保守的机制。所描述的方法将受益于转基因18、22</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 el minatio de economia y v尔蒂维达德 (mineco) 的赠款的支持;由欧洲区域发展基金 (erdf) 共同资助的: m. g. f. fuortes 纪念研究金、stephen w. kuffler 研究金基金、laura wlaura 捐赠夏季研究金基金颁发的竞争性研究奖, fischbach 研究金, 以及海洋生物实验室的大一代基金和国家 xenopus 资源 RRID:SCR_013731 (伍兹霍尔, 马萨诸塞州), 在那里进行了这项工作的一部分。我们还感谢 cerca proplit/generalitit发电量 de catalunya 的机构支持。a. l. 是塞拉·胡恩特的同伴。
Materials
| Salts for aquariums (Instant Ocean Salt) | Tecniplast | XPSIO25R | |
| Tricaine (Ethyl 3-aminobenzoate methanesulfonate) | Sigma-Aldrich | E10521 | |
| Tweezers #5 (tip 0.025 x 0.005 mm) | World Precision Instruments | 501985 | |
| Vannas Scissors (tip 0.015 x 0.015) | World Precision Instruments | 501778 | |
| Whatman qualitative filter paper | Fisher Scientific | WH3030917 | |
| X. laevis tubb2-GFP | National Xenopus Resource (NXR), RRID:SCR_013731 | NXR_0.0035 | |
| X.tropicalis NBT-GFP | European Xenopus Resource Center (EXRC) RRID:SCR_007164 | ||
| CellTracker CM-DiI | ThermoFisher Scientific | C-7001 | |
| Calcium Green dextran, Potassium Salt, 10,000 MW, Anionic | ThermoFisher Scientific | C-3713 | |
| Borosilicate capillaries for microinjection | Sutter Instrument | B100-75-10 | O.D.=1.0 mm., I.D.=0.75 mm. |
| Puller | Sutter Instrument | P-97 | |
| Microinjector | Parker Instruments | Picospritzer III | |
| Sylgard-184 | Sigma-Aldrich | 761028-5EA | |
| Microfil micropipettes | World Precision Instruments | MF28G-5 | |
| Upright microscope | Zeiss | AxioImager-A1 | |
| Master-8 stimulator | A.M.P.I. | ||
| CCD Camera | Hamamatsu | Image EM | |
| Solenoid valves | Warner Instruments | VC-6 Six Channel system | |
| Dow Corning High Vacuum Grease | VWR Scientific | 636082B | |
| Tubocurarine hydrochloride | Sigma-Aldrich | T2379 | |
| CCD Camera | Zeiss | MRC-5 Camera | Controlled by Zen software |
| camera lens | Thorlabs | MVL8ML3 | There are multiple possibilities that should be adapted to the camera model used |
| Epoxy resin | RS Components | ||
| Manifold | Warner Instruments | MP-6 perfusion manifold | |
| Micromanipulator for local delivery of solutions | Narishige | MN-153 | |
| Mini magnetic clamps | Warner Instruments | MAG-7, MAG-6 | |
| Polyethylene tubing | Warner Instruments | 64-0755 | O.D.=1.57 mm., I.D.=1.14 mm. |
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Cite This Article
Terni, B., Pacciolla, P., Perelló, M., Llobet, A. Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles. J. Vis. Exp. (142), e58028, doi:10.3791/58028 (2018).