运动神经元细胞外识别的肌肉运动游泳池<em>海兔夜蛾</em
Summary
在大确定的神经元（动物<em>例如，</em>软体动物），分析细胞内的技术是通过使用电机池<sup> 1,2,3,4</sup>。最近，我们开发出一种技术，细胞外刺激和记录单个神经元<em>海兔夜蛾</em<sup></sup>。现在我们描述了使用这种技术来唯一地识别和表征运动神经元内的电机池的协议。
Abstract
在动物与大确定的神经元（ 例如软体动物），分析电机池在使用细胞内的技术1,2,3,4。最近，我们开发了一种技术，细胞外刺激，记录单个神经元在海兔夜蛾 5。现在我们描述了使用这种技术来唯一地识别和表征运动神经元内的电机池的协议。
此外技术方面具有优势。首先，细胞外电极可以刺激并记录神经元通过鞘5，所以，它并不需要被删除。因此，神经元将健康的细胞外实验，在细胞内的。其次，如果神经节是由适当的钉扎的鞘的旋转，外电极可以访问神经元的神经节上的两侧，这使得它更容易和更有效地识别多个神经元中的相同的制备。三，extracellu拉尔电极不需要穿透细胞，从而可以很容易地来回移动，神经元之间，他们造成损伤少。这是非常有用的，当一个人试图记录多个神经元在重复的运动模式，可能只持续几分钟。四，外电极是在肌肉运动更灵活，比细胞内的。细胞内的电极可以拉出来，破坏肌肉收缩的神经元。相反，由于外电极被轻轻地按压到上述鞘神经元，它们通常留上面相同的神经元在肌肉的收缩，因此可以用在更完整制剂。
为了唯一地识别的用于电动机池（尤其是，I1/I3在海兔肌肉）使用细胞外电极的运动神经元，一个可以使用的功能，不需要标准：细胞内测量胞体的大小和位置，轴突投射，和肌肉的神经支配4[6,7]。对于使用特定电机池来说明的技术，我们记录从颊神经测量轴突突起2和3，并测定I1/I3肌肉的收缩力的确定的图案为个别的运动神经元的肌肉的神经支配。
我们证明首先确定运动神经元肌肉神经支配的完整过程，然后描述他们的时间在运动模式，创建一个简单的诊断方法快速鉴定。简化的和更快速的诊断方法是优越的更完整的准备工作， 例如 ，在暂停的的口腔大量制备8 或 9 体内 。这个过程也可以被应用于其它电机池10,11,12在海兔或其他动物系统中2,3,13,14。
Protocol
1。准备录音碟力传感器实验中，颊神经节，神经节，和颊质量被放置在一个圆形的高硼硅菜是专门为力研究。 为了诱导摄食状花纹，在实验中，我们需要运用非水解胆碱能激动剂氨甲酰胆碱的神经节15。以避免直接接触到颊神经节和颊质量由氯化氨甲酰胆碱，单独的腔室所需的隔离颊神经节的神经节和颊质量（ 图1）。 由于颊质量远厚于颊神经节，它们…
Representative Results
图4和图5结果显示了典型的用于识别两个I1/I3运动神经元。期间egestive等和摄食状的图案（ 图4C，4D），图4示出了一个大的运动神经元，B3，苏摩录音。一个一个对应的尖峰上的SOMA通道和同侧的BN2通道（ 图4E）的特异性的B3 SOMA记录的过程中保持模式。 B3火灾过程中的中，后期的收缩阶段的模式。从图4和其他的结果（图中未示出）中，…
Discussion
典型的做法是在动物与大确定的神经元，如软体动物（例如， 椎实螺 ， 螺旋 ，和海兔 ），分析电机池使用细胞内记录1,2,3,4。在这个协议中，我们描述了一个过程，用于唯一地识别用于电动机的运动神经元池使用一种细胞外的技术。作为一个例子，这个过程中，我们使用的测力。也可以使用肌电图测量肌肉的神经支配。简要地说，这样做的，该协议附加钩电极I1/I3肌…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国国立卫生研究院授予NS047073和国家科学基金会资助DMS1010434。
Materials
| Name | Company | Catalog Number | Comments |
| Sodium chloride | Fisher Scientific | S671 | Biological, Certified |
| Potassium chloride | Fisher Scientific | P217 | Certified ACS |
| Magnesium chloride hexahydrate | Acros Organics | 19753 | 99% |
| Magnesium sulfate heptahydrate | Fisher Scientific | M63 | Certified ACS |
| Calcium chloride dihydrate | Fisher Scientifc | C79 | Certified ACS |
| Glucose (dextrose) | Sigma-Aldrich | G7528 | BioXtra |
| MOPS buffer | Acros Organics | 17263 | 99% |
| Carbachol | Acros Organics | 10824 | 99% |
| Sodium hydroxide | Fisher Scientific | SS255 | Certified |
| Hydrochloric acid | Fisher Scientific | SA49 | Certified |
| Single-barreled capillary glass | A-M Systems | 6150 | |
| Flaming-Brown micropipette puller model P-80/PC | Sutter Instruments | Filament used: FT345B | |
| Enamel coated stainless steel wire | California Fine Wire | 0.001D, coating h | |
| Household Silicone II Glue | GE | ||
| Duro Quick-Gel superglue | Henkel corp. | ||
| A-M Systems model 1700 amplifier | A-M Systems | Filter settings: 10-500 Hz for the I2 nerve/muscle; 300-500 Hz for all the other nerves | |
| Pulsemaster Multi-Channel Stimulator | World Precision Instruments | A300 | |
| Stimulus Isolator | World Precision Instruments | A360 | |
| AxoGraph X | AxoGraph Scientific | Software for recordings | |
| Gold Connector Pins | Bulgin | SA3148/1 | |
| Gold Connector Sockets | Bulgin | SA3149/1 | |
| Sylgard 184 Silicone Elastomer | Dow Corning | ||
| 100 x 15 mm Crystalizing Dish | Pyrex | ||
| High Vacuum Grease | Dow Corning | ||
| Pipet Tips | Fisher Scientific | 21-375D | |
| Minutien Pins | Fine Science Tools | 26002-10 | |
| Modeling Clay | Sargent Art | 22-4400 | |
| Whisper Air Pump | Tetra | 77849 | |
| Aquarium Tubing | Eheim | 7783 | 12/16 mm |
| Elite Airstone | Hagen | A962 | |
| Vannas Spring Scissors | Fine Science Tools | 15000-08 | |
| Dumont #5 Fine Forceps | Fine Science Tools | 11254-20 | |
| Kimwipes | Kimberly-Clark | 34155 |
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Citer Cet Article
Lu, H., McManus, J. M., Chiel, H. J. Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica. J. Vis. Exp. (73), e50189, doi:10.3791/50189 (2013).