电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统
Summary
迅速<em>在体内</em>分析测试neuromodulatory使用巨人光纤系统（GFS）的化合物<em>果蝇</em>描述。 nanoinjections动物的头，随着政府飞行服务队的电生理记录可以揭示生物活性化合物对神经或肌肉。
Abstract
体内活性筛选化合物可以用来作为第一步，以确定可能到1,2药物制剂开发的候选人。我们开发了一种新型的nanoinjection /电检测，使检测的神经电路介导的在果蝇 3,4逃生响应功能的生物活性化合物的调节作用。我们在体内实验中，使用果蝇巨纤维系统（GFS， 图1）允许不同类型的化合物，如小分子肽的筛选，只需要最小数量引起的效果。此外，果蝇的飞行服务队提供了一个潜在的分子靶点神经或肌肉的种类繁多。 （GFS）巨纤维突触电（峡路口）以及化学（胆碱）到外围的中间神经突触（PSI）和“Tergo Trochanteral肌肉神经元（TTMn）5 </SUP>。背侧纵肌神经元DLMn（）连接的PSI是依赖Dα7烟碱乙酰胆碱受体（nAChRs）的6。最后，神经肌肉接头（NMJ）的TTMn和DLMn跳（TTM）和飞行肌（DLM），是谷氨酸7-12。在这里，我们演示了如何注入化合物的纳升数量级，而获得巨人光纤系统，以及如何监察该电路的功能复合的影响的电生理细胞内记录。我们显示枸橼酸methyllycaconitine（MLA）的，胆碱受体拮抗剂，破坏PSI的DLMn连接，但不能连接或跳跃或飞行肌肉的功能NMJ的GF TTMn的的的特异性。
在这部影片开始之前，它是至关重要的，你仔细地观察和熟悉的朱庇特的视频名为“电生理记录，从四melan巨纤维通路“ogaster从奥古斯丁等 7，视频这里的目的是为这一现有技术的扩展。在这里，我们使用的细节，只对另外的的配对nanoinjections和监测技术的电生理记录方法和重点。
Protocol
1。电钻机设置详细描述了为电钻机设置所需的设备由奥古斯丁等 。在该杂志14。为所需的电生理仪器的详细解释，请参阅本文。 加入了第六的微操作机器人，其持有的nanoinjector修改先前所描述的电钻机设置14。为方便应置于两者之间的刺激电极显微操作，在图2所示的动物的头。 在实验开始前，确保你有一个旋转轴与所有显微操作的舒适?…
Discussion
这里介绍的nanoinjection /电生物活性，可快速筛选的化合物在果蝇的神经系统。这是一个小说在体内的技术，需要少量的化合物，在一个特点的神经电路引起的各种分子靶点的作用。这种方法可以用来测试不同的化合物的生物活性，从未知毒素市售药物制剂。
在这里，我们证明了我们的检测功能，使用工作重点，其中有巨人光纤系统（GFS）的果蝇（ 图5）效?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要承认在马里实验室和Godenschwege的实验室，特别是艾琳米泽成员，征求意见，并帮助这个协议。这项工作是由国立神经疾病研究所和中风授予R21NS06637 FM和TAG; AB公司是由国家科学基金会奖号码082925，URM的资助：为未来的研究人员结合生物学。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Recording glass electrodes: borosilicate glass capillaries | World Precision Instruments | 1B100F-4 | 1.0mm OD, 0.58mm ID |
| Stimulator | Grass Instruments | Model S48 | |
| Amplifier | Getting Instruments Inc. | Model 5A | |
| Data acquisition Software: Digidata | Molecular Devices | Model 1440A | |
| Data collection software: pCLAMP | Molecular Devices | Version 10 | |
| Stereomicroscope with fiber optic microscope ring illuminator | AmScope | SM-4T Model HL250-AR |
|
| Dissecting scope for mounting | AmScope | SM-2TZ | |
| Kite Manual Micromanipulator & Tilting Base | World Precision Instruments | Model # M3301 Kite: Model # KITE-M3-L |
|
| Drosophila melanogaster Wild 10E genotype (wild type strain) | Bloomington Stock center | Stock # 3892 | |
| Vertical pipette puller | David Kopf Instruments | Model 700c | |
| Injection glass micropipettes: Borosilicate glass capillaries | World Precision Instruments | Catalogue # 4878 | 1.14mm OD, 0.5mm ID |
| Silicon oil | Fisher | Catalogue # S159-500 | |
| Beveler | Sutter Instrument Co. | K.T. Brown Type Model # BV-10 |
|
| Nanoliter2000 | World Precision Instruments | Catalogue # B203XVY | |
| Blue food coloring | McCormick | N/A | Ingredients: Water, Propylene Glycol, FD&C Blue 1, and 0.1% Propylparaben (preservative). |
| Methyllycaconitine citrate (MLA) | Tocris Bioscience | Catalogue # 1029 | |
| Plastic wax sticks | Hygenic Corporation (Akron Ohio USA) |
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Tags
NanoinjectionElectrophysiology AssayScreenBioactivityCompoundsDrosophila MelanogasterGiant Fiber SystemIn Vivo ActivityPharmacological AgentsModulatory EffectsNeuronal CircuitSmall MoleculesPeptidesMolecular TargetsGap JunctionsCholinergicNicotinic Acetylcholine ReceptorsNeuromuscular JunctionsGlutamatergicNanoliter QuantitiesIntracellular Recordings
Cite This Article
Mejia, M., Heghinian, M. D., Busch, A., Marí, F., Godenschwege, T. A. Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System. J. Vis. Exp. (62), e3597, doi:10.3791/3597 (2012).