从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录
Summary
在果蝇第三龄幼虫神经肌肉交界处, 可从可视化突触 boutons 局部记录突触电流。这种技术可以监测单个突触溥的活动。
Abstract
果蝇神经肌肉结 (NMJ) 是研究谷氨酸突触传递的一个很好的模型系统。我们描述了从可视化 boutons 在果蝇幼虫 NMJ 的突触电流的焦 macropatch 记录技术。这项技术需要定制制作记录 micropipettes, 以及一个复合显微镜, 配备了高倍率, 长距离水浸泡物镜, 差分干扰对比 (DIC) 光学, 和荧光附件.记录电极定位在选定的突触溥的顶部, 与 DIC 光学, epi 荧光, 或两者。这种技术的优点是它允许监测有限数量的释放点的突触活动。该记录电极的直径为几微米, 而位于电极外缘外的释放位置不会对记录的电流产生显著影响。记录的突触电流具有快速的动力学, 可以很容易地解决。这些优势是特别重要的研究突变飞线与增强自发或异步突触活动。
Introduction
果蝇是研究控制突触传递的分子机制的一个很好的模型系统。在果蝇的神经肌肉系统是谷氨酸, 因此,果蝇神经肌肉结 (NMJ) 可以用来研究谷氨酸释放的保守特征。自1月和1月的研究1, 第三龄幼虫已广泛用于研究诱发和自发突触传播通过监测兴奋性结电位 (EJPs) 或电流 (EJCs)。EJPs 通常记录细胞与一个尖锐的玻璃微电极, 它们反映了整个 NMJ 的活动, 包括所有的 boutons 在给定的肌肉纤维的突触。
相比之下, 通过在神经元终端或突触静脉曲张附近定位一个微尖端, 可以记录局部的少量释放点的活动。这项技术最初受聘于 Katz 和 Miledi2, 并成功地使用了一些 NMJ 准备, 包括青蛙3,4,5, 鼠标6,7,8, 甲壳动物9,10,11,12,13,14,15,16, 和果蝇17,18,19,20,21,22,23。这一方法进一步开发了 Dudel, 谁优化 macropatch 重新编码电极24,25。在 Dudel 的实现中, 此技术与松散膜片钳方法26紧密匹配。
果蝇幼虫 NMJ 已明确定义突触 boutons, 和基因编码的神经荧光标记的转基因品系 (见材料表) 是现成的。这些优势使我们能够记录 EJCs 和 mEJCs 从一个选定的突触溥20,21,22。在这里, 我们详细描述这一技术。
Protocol
1. 制作记录电极 拉动玻璃电极 使用以下协议为微电极拉出器 (参见 材料表 ): 1 行: 热510拉速30时间 250;2行: 热490拉速30时间 250. 注: 时间单位对应于每单位0.5 毫秒;其他单位是相对的。在进行坡道试验后, 应调整每根灯丝的热量值. 使用显微镜 (35x 放大倍数) 来确保所拉电极的内径在 7-10 和 #181 的范围内; m (…
Representative Results
焦点 macropatch 录音可以监视所选突触 boutons 的突触活动 (图 5)。当电极位于突触溥 (图 5A, 站点 1) 的顶部时, 记录的 mEJCs (图 5C, 站点 1) 的振幅明显超过噪音水平和尖锐的上升阶段 (在毫秒范围内)。当记录电极被移离突触溥的几微米 (图 5A, 站点 2) 时, 记录的 mEJCs 的振幅几乎下降到噪音…
Discussion
果蝇代表了研究突触传递的一个有利的模型有机体。在幼体 NMJ 使用了几种记录配置, 包括突触电位的胞内记录、两个电极电压钳33、34和焦点 macropatch 的突触电流记录。在这里描述的突触电流的录音。后一种技术允许精确定量的突触传输在可视化的 boutons。
所描述的协议的成功与否, 关键取决于是否有能力清楚地直观地显示感…
Divulgations
The authors have nothing to disclose.
Acknowledgements
由 NIH 资助 R01 MH 099557
Materials
| Sutter P-97 | Sutter instrument | P-97 | Microelectrode puller |
| Narishige MF-830 | Narishige | MF-830 | Microforge |
| WPI MF200 | WPI | MF200 | Microforge |
| Glass capilaries | WPI | B150-86-10 | Glass capilaries |
| Microtorch 1WG61 | Grainer | 1WG61 | Microtorch |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | SYLGARD 184 | Silicone for dissection plates preparation |
| Dissection pins | Amazon | B00J5PMPJA | Pins for larvae positioning |
| Tweezers | WPIINC | 500342 | Tweezers for placing pins, removing the guts and tracheas. |
| Scissors | WPIINC | 501778 | Scissors for cutting the cuticula of the larvae and nerves. |
| Olympus BX61WI | Olympus | BX61WI | Upright microscope |
| Olympus Lumplan FL N 60x | Olympus | UPLFLN 60X | Microscope objective 60X |
| Olympus UPlan FL N 10x | Olympus | Uplanfl N 10X | Microscope objective 10X |
| Narishige Micromanipulator | Narishige | MHW-3 | Three-axis Water Hydraulic Micromanipulator |
| npi Electronic GmbH ELC-03XS | npi Electronic GmbH | ELC-03XS | Electrophysiological amplifier |
| A.M.P.I Master 8 | A.M.P.I. | Master 8 | Electrical stimulator |
| A.M.P.I Iso-Flex | A.M.P.I. | Iso-Flex | Stimulus isolator |
| TMC antivibration table | TMC | 63-9090 | Antivibration table |
| TMC Faraday cage | TMC | 81-333-90 | Faraday cage |
| Digidata 1322A | Axon Instruments | Digidata 1322A | Digidata |
| Computer | Dell | Dell Dimension 5150 | Computer with Win XP OS |
| Electrode holder | WPI | MEH3SW | Electrode holder |
| Optical filter | Omega optical | XF 115-2 | Filter cube for Green Fluorescent Protein (GFP) detection |
| pCLAMP 8 | Axon Instruments | 8.0.0.81 | Software for signal recording |
| Quantan | In-house software | – | Software for signal processing |
| Canton-S (Wildtype) | Bloomington Stock Center | 64349 | Control fly line |
| cpx SH1 | Generous Gift of J.T. Littleton | – | Complexin knock-out fly line with increased spontaneous exocytosis |
| CD8-GFP | Bloomington Stock Center | 5137 | Fly line with neuronal fluorescent (GFP) Tag |
References
- Jan, L. Y., Jan, Y. N. Properties of the larval neuromuscular junction in Drosophila melanogaster. J Physiol. 262 (1), 189-214 (1976).
- Katz, B., Miledi, R. The effect of temperature on the synaptic delay at the neuromuscular junction. J Physiol. 181 (3), 656-670 (1965).
- Macleod, G. T., Gan, J., Bennett, M. R. Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals. J Neurophysiol. 82 (3), 1133-1146 (1999).
- Macleod, G. T., Farnell, L., Gibson, W. G., Bennett, M. R. Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus). J Neurophysiol. 81 (3), 1135-1146 (1999).
- Zefirov, A., Benish, T., Fatkullin, N., Cheranov, S., Khazipov, R. Localization of active zones. Nature. 376 (6539), 393-394 (1995).
- Macleod, G. T., Lavidis, N. A., Bennett, M. R. Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens. J Physiol. 480 (Pt 1), 61-70 (1994).
- Samigullin, D., Bill, C. A., Coleman, W. L., Bykhovskaia, M. Regulation of transmitter release by synapsin II in mouse motor terminals. J Physiol. 561 (Pt 1), 149-158 (2004).
- Coleman, W. L., Bykhovskaia, M. Rab3a-mediated vesicle recruitment regulates short-term plasticity at the mouse diaphragm synapse. Mol Cell Neurosci. 41 (2), 286-296 (2009).
- Atwood, H. L., Parnas, H., Parnas, I., Wojtowicz, J. M. Quantal currents evoked by graded intracellular depolarization of crayfish motor axon terminals. J Physiol. 383, 587-599 (1987).
- Parnas, H., Dudel, J., Parnas, I. Neurotransmitter release and its facilitation in crayfish. I. Saturation kinetics of release, and of entry and removal of calcium. Pflugers Arch. 393 (1), 1-14 (1982).
- Wojtowicz, J. M., Marin, L., Atwood, H. L. Activity-induced changes in synaptic release sites at the crayfish neuromuscular junction. J Neurosci. 14 (6), 3688-3703 (1994).
- Zucker, R. S. Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarizing pulses. J Physiol. 241 (1), 69-89 (1974).
- Zucker, R. S. Changes in the statistics of transmitter release during facilitation. J Physiol. 229 (3), 787-810 (1973).
- Worden, M. K., Bykhovskaia, M., Hackett, J. T. Facilitation at the lobster neuromuscular junction: a stimulus-dependent mobilization model. J Neurophysiol. 78 (1), 417-428 (1997).
- Bykhovskaia, M., Hackett, J. T., Worden, M. K. Asynchrony of quantal events in evoked multiquantal responses indicates presynaptic quantal interaction. J Neurophysiol. 81 (5), 2234-2242 (1999).
- Bykhovskaia, M., Polagaeva, E., Hackett, J. T. Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse. Brain Res. 1019 (1-2), 10-21 (2004).
- Cooper, R. L., Stewart, B. A., Wojtowicz, J. M., Wang, S., Atwood, H. L. Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus. J Neurosci Methods. 61 (1-2), 67-78 (1995).
- Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J., Wu, C. F. Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions. J Comp Physiol A. 175 (2), 179-191 (1994).
- Pawlu, C., DiAntonio, A., Heckmann, M. Postfusional control of quantal current shape. Neuron. 42 (4), 607-618 (2004).
- Akbergenova, Y., Bykhovskaia, M. Synapsin maintains the reserve vesicle pool and spatial segregation of the recycling pool in Drosophila presynaptic boutons. Brain Res. 1178, 52-64 (2007).
- Akbergenova, Y., Bykhovskaia, M. Enhancement of the endosomal endocytic pathway increases quantal size. Mol Cell Neurosci. 40 (2), 199-206 (2009).
- Vasin, A., Volfson, D., Littleton, J. T., Bykhovskaia, M. Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion. Biophys J. 111 (9), 1954-1964 (2016).
- Wong, K., Karunanithi, S., Atwood, H. L. Quantal unit populations at the Drosophila larval neuromuscular junction. J Neurophysiol. 82 (3), 1497-1511 (1999).
- Dudel, J. The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction. Pflugers Arch. 391 (1), 35-40 (1981).
- Dudel, J. Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle. Pflugers Arch. 422 (2), 129-142 (1992).
- Marrero, H. G., Lemos, J. R. . Loose-Patch-Clamp method. , (2007).
- Wu, W. H., Cooper, R. L. Physiological recordings of high and low output NMJs on the crayfish leg extensor muscle. J Vis Exp. (45), (2010).
- Verstreken, P., Ohyama, T., Bellen, H. J. FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction. Methods Mol Biol. 440, 349-369 (2008).
- Brent, J. R., Werner, K. M., McCabe, B. D. Drosophila larval NMJ dissection. J Vis Exp. (24), (2009).
- Imlach, W., McCabe, B. D. Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae. J Vis Exp. (24), (2009).
- Bykhovskaia, M. Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN. J Neurosci Methods. 168 (2), 500-513 (2008).
- Huntwork, S., Littleton, J. T. A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth. Nat Neurosci. 10 (10), 1235-1237 (2007).
- Zhong, Y., Wu, C. F. Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade. Science. 251 (4990), 198-201 (1991).
- Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire. Neuron. 28 (3), 941-953 (2000).
- Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. T. Spontaneous and evoked release are independently regulated at individual active zones. J Neurosci. 33 (44), 17253-17263 (2013).
Citer Cet Article
Vasin, A., Bykhovskaia, M. Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction. J. Vis. Exp. (127), e56493, doi:10.3791/56493 (2017).