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 JoVE Biology

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

1, 1, 1

1Department of Biology, Brandeis

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    Summary

    此过程使用蓝色光激活藻通道和细胞特异性基因表达的工具,以唤起光脉冲在神经肌肉接头(NMJ)突触潜力

    Date Published: 3/16/2009, Issue 25; doi: 10.3791/1133

    Cite this Article

    Hornstein, N. J., Pulver, S. R., Griffith, L. C. Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions. J. Vis. Exp. (25), e1133, doi:10.3791/1133 (2009).

    Abstract

    Protocol

    第1部分:动物保健和遗传杂交

    1. 保持无人机,ChR28个单独的瓶含有标准的飞行媒体OK371 GAL4飞线。
    2. 收集OK371 - GAL4飞线和UAS - ChR2线的男性处女。
    3. 将男性和女性在包含标飞1毫米全反式视网膜(ATR)的混合介质的小瓶。 ATR食品应首先融化在定期飞〜1分钟微波介质。一旦融化,使冷却约30秒到一分钟,然后加入100μL100%乙醇10毫升每飞行媒体的ATR 100毫米。瓶置于冰上,然后将其存储在暗区,在4 ° C。
    4. 让果蝇交配打下鸡蛋中的暗区,在22-25 ° C。
    5. 等待3-4天,直到3龄幼虫可见,在这一点上,处理成蝇。

    第2部分:钻机安装

    1. 附加任何10X解剖范围眼睛一块蓝色LED(在我们的例子中,卡尔蔡司公司,www.zeiss.com,Thornwood,纽约),带散热片的光源(托尔实验室,www.thorlabs.com,牛顿,新泽西州)。附加磁性底座后,钳。电气接地屏蔽,以减少由光源所产生的电气噪声覆盖散热片。
    2. 对空气电钻机表光源放置磁性底座。
    3. 光源连接到控制电路和控制电路连接到外部电压源(4 / 30 PowerLab系统,ADInstruments,www.adinstruments.com,科罗拉多州的斯普林斯,一氧化碳)。
    4. 给1-5 V脉冲控制电路,以激活蓝灯。
    5. 调整磁性底座和光源,直到蓝色光柱集中幼虫清扫占用的面积。

    第3部分:夹层

    1. 放置六个0.1毫米到地板的一个sylgard内衬的菜虫引脚。
    2. 删除任何塑料的Petri盘3 从食物中媒体和地方的幼虫。
    3. 用生理盐水冲洗,清除食物的幼虫。
    4. 幼虫在解剖盘附近的拉引脚和补充生理盐水半水平。
    5. 东方的幼虫,所以,你可以看到两个银色管(气管),沿动物的背水面上运行。
    6. 尾巴大针直接插入气管导管之间的。保持幼虫和其头部放入第二个大针,一定要纵向舒展身体。
    7. 设为尾巴附近的一个小切口,并继续它的身体的长度。确保剪刀的技巧提高,使不意外削减腹侧神经和/或体壁肌肉。
    8. 动物的四个角上放置四个引脚现在开放的腹部。他们解剖盘设置成圆角动物。引脚编制出教。
    9. 使用镊子和剪刀,消除动物的内脏,气管和脂肪组织。
    10. 用生理盐水冲洗准备。
    11. 找到额叶(图A)和腹侧神经节的预习。
    12. 采用微型剪刀,小心地穿过仅次于额叶腹侧神经节。

    第4部分:肌肉录音和蓝色光刺激

    1. 拉10-20μω电极使用电子电极拉马(萨特仪器)。
    2. 填写带3米氯化钾拉电极。
    3. 广场充满了附近幼虫准备在解剖显微镜上电钻机下的显微电极,在电极支架和机动电极尖端。
    4. 确定在任何体壁段的肌肉(图A)6(M6)。
    5. 小心地放入M6的电极,并观看快速极化。肌肉静息膜电位,应该是任何地方从-30毫伏到-70毫伏。
    6. 调整蓝色的光芒,使解剖准备在光束中心。
    7. 给予20-100毫秒脉冲电压控制电路。逐步增加提供给控制电路的电压。注意在肌肉细胞的兴奋交界的潜力(图1B)。

    代表性的成果:

    图1A显示了录音的设置和剔骨准备的示意图。图1B显示了典型的EJP短的光脉冲诱发。 EJP幅度显示总结既支配M6从两个电机神经元的幅度。光照强度下,只激活一个运动单位(数据未显示)。

    图1

    图1:A)细胞内记录钻机和蓝色LED的一般原理图。拆下来抑制大脑(BR)是在腹侧神经节(VG)的有节奏的活动。 ChR2表示使用GAL4 - UAS的系统在电机神经元。二)从M6的肌肉细胞内记录。 40毫秒蓝色光脉冲(127μW/毫米2)可靠地唤起M6突触潜力大(星号) 。

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    Discussion

    关键步骤涉及的初步解剖和进入肌肉细胞。如果神经被切断,或在初始剥离肌肉受损,就很难继续实验的休息。在清扫过程中,必须非常谨慎的角度在背的切口,向上为尽可能解剖剪刀。在第二个关​​键的一步,进入肌肉细胞,一个必须注意的一个极化过去〜30 mV的。 -30毫伏以上的值指示电极是不是肌肉细胞内或在一个不健康的细胞正常。

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    Disclosures

    我们有没有利益冲突披露。

    Acknowledgements

    这项工作得到了国家卫生赠款RO1GM - 33205和MH - 067284研究院立法会格里菲斯和一个布兰代斯大学的夏季本科生科研奖学金,新泽西州Hornstein。作为2008年神经系统和行为暑期课程(镍氢补助金:R25 MH059472)的一部分,马在伍兹霍尔海洋生物实验室进行这项技术的初步实验。

    Materials

    Name Company Catalog Number Comments
    Sylgard Ellsworth Adhesives 4019862 www.ellsworth.com
    Minutens Pins Fine Science Tools 26002-10 www.finescience.com
    Dissecting Dish Fisher Scientific www.fishersci.com
    Neuroprobe Intracellular Amplifier and Head Stage A-M Systems 680100 www.a-msystems.com
    Powerlab 4/30 data acquisition system ADInstruments www.adinstruments.com
    Grass stimulator Grass Technologies www.grasstechnologies.com
    Desktop Computer Dell www.dell.com
    Dissecting Scope Leica Microsystems www.leica-microsystems.com
    Light Source Dolan-Jenner Industries 41446-062 www.dolan-jenner.com
    Fly Media
    All-Trans-Retinal Sigma-Aldrich 116-31-4 www.sigmaaldrich.com
    OK-371 Gal4 Flies Bloomington Stock center
    UAS-ChR2 Flies Fiala lab, Griffith lab
    LED controller circuit Built in Griffith lab http://www.ledsupply.com
    http://www.futureelectronics.com
    Composed of:
    1. 200 mA Buck Puck
    2. Blue LED
    3. Insulated wire
    4. Circuit bread board
    LED Heat Sink Thorlabs Inc. http://www.thorlabs.com/
    Air Table TMC http://www.techmfg.com/products/accessories/intro3.html
    Faraday Cage Built in Griffith lab
    Leica Leitz M Micro-Manipulator Leica Microsystems ACS01 www.leica-microsystems.com
    Electrode Holder Axon Instruments www.axon.com
    Borosilicate Glass FHC, Inc. www.fh-co.com/p14-15.pdf
    Electrode Puller Sutter Instrument Co. www.sutter.com
    HL 3.1 Saline with 0.8mM Ca2+ Contents (mM):
    NaCl:70 KCl:5 CaCl2: 0.8
    MgCl2:4Sucrose:115
    NaHCO3: 10 Trehalose: 5 HEPES
    Micro-Dissection Tools Fine Science Tools www.finescience.com

    References

    1. Keshishian, H., Broadie, K., Chiba, A., and Bate, M., The Drosophila neuromuscular junction: a model system for studying synaptic development and function. Annu Rev Neurosci 19, 545 (1996);
    2. Collins, C. A. and DiAntonio, A., Synaptic development: insights from Drosophila. Curr Opin Neurobiol 17 (1), 35 (2007);
    3. Lagow, R. D. et al., Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion. PLoS Biol 5 (4), e72 (2007).
    4. Nagel, G. et al., Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol 15 (24), 2279 (2005);
    5. Nagel, G. et al., Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci U S A 100 (24), 13940 (2003);
    6. Schroll, C. et al., Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae. Curr Biol 16 (17), 1741 (2006).
    7. Lin, D. M., Auld, V. J., and Goodman, C. S., Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers. Neuron 14 (4), 707 (1995).
    8. Greenspan, Ralph, Fly Pushing: The Theory and Practice of Drosophila Genetics, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2004).

    Comments

    1 Comment

    Hi Nicholas,

    A nice vido. I want to konw what model of light source did you use ? I could not find it by the Cata Num on the website.

    Thanks a lot.

    Kewen

    School of medicine Zhejiang University
    Reply

    Posted by: Jiang K.December 17, 2009, 9:02 PM

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