一个<em在体外</em制备与生理变动，为激发和记录进给电机程序<em>海兔夜蛾</em
Summary
我们描述了一种技术，细胞外记录和刺激神经，肌肉和个人的神经元，<em在体外</em>同时引出和观察在馈送装置的取食行为的不同类型的<em>海兔</em>。
Abstract
多功能的，一个外围结构的能力，生成多个不同的行为，使动物能够迅速调整自己的行为，以适应不断变化的环境。海洋软体动物 Aplysia 夜蛾提供了一个易于处理系统的多功能性的研究。 海兔的饲养过程中，产生了几种不同类型的使用相同的进给装置，颊质量的行为。控制这些行为的神经节包含了一些大型，确定了神经元的电生理研究访问。已经描述了这些神经元的活性，可分为两种类型，摄食和egestive方案上的神经活动的定时关闭食品抓取器相对的神经活动，是根据在电机的程序，protracts或缩回抓钳2。然而，在孤立的神经，肌肉的运动，会产生这些行为是不存在的，使得它更难某些电机的方案观察是否是相关的实际行为。 在体内 ，神经和肌肉的录音中已取得相应的喂养方案2,3,4，但它是非常困难的直接记录从单个神经元的5。此外， 在体内 ，摄食方案可进一步分为叮咬和燕子1,2，区别是难以在体外准备。
暂停的的口腔大量制备（ 图1）孤立的神经节和完整的动物之间架起了一座桥梁。在该制剂中，可摄食行为-包括咬和吞咽-和egestive的行为（拒绝）引出，在同一时间可以记录作为单独的神经元，刺激使用胞外电极6。与这些不同的行为的进给运动，可以收音DED，量化，直接关系到电机的程序。运动程序中的悬浮口腔大量制备似乎是更相似， 在体内观察到的是运动程序引起的孤立节。因此，电动机在此制备方案可以更直接地在体内的行为有关的，在相同的时间，单个神经元的较完整的动物更容易访问的记录和刺激。此外，作为隔离神经节和完整的动物之间的中间步骤，从暂停的颊质量结果可以有助于更加减少，更完整的设置中获得的数据的解释。暂停的的口腔大量制备是一个非常有用的工具，在海兔的神经控制的多功能特性。
Protocol
1。溶液的制备为了制备氯化镁溶液是等渗的海水，其中动物保持（〜1,000 millosmolar），在所需的量的水平的一大罐马克。这个水平的约80％的蒸馏水填充的罐体，并创建333 mM的溶液的最终体积中称出适量的氯化镁六水合物。添加氯化镁的酒壶，盖上盖子，用力摇晃，直到氯化镁完全溶解，然后加入蒸馏水，直至达到最终体积。 为了制备海兔生理盐水，再次马克一大罐的所需?…
Representative Results
When an extracellular electrode is positioned above a neuron’s soma and used to stimulate the neuron, a one-for-one correspondence between spikes on the soma channel and on the nerve(s) the neuron projects to can be observed (Figure 6, left panel, stimulation of identified neuron B9). The soma channel (top channel) is set to stimulating mode when the current is applied (time 1 in the figure), and is then quickly switched to recording mode (time 2). By maintaining the position of the electrode, the…
Discussion
以前的工作特点的海兔电机计划在完整的动物和减少的准备工作，如孤立的神经。在完整的动物，虽然单个神经元的记录已经获得了5，这样的实验是非常困难的，和电极不能移动从神经元到神经元在喂养过程中。在离体神经节，神经活动引起的进给运动不能观察到。暂停的的口腔大量制备这两个极端之间架起了一座桥梁。
其他半完整制剂已被用于在以前的研?…
Disclosures
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: 300-500 Hz nerves,10-500 Hz I2 muscle | |
| Pulsemaster Multi-Channel Stimulator | World Precision Instruments | A300 | |
| Stimulus Isolator | World Precision Instruments | A360 | |
| AxoGraph X | AxoGraph Scientific | ||
| Veeder-Root Totalizing Counter | Danaher | C342-0562 | |
| Gold Connector Pins | Bulgin | SA3148/1 | |
| Gold Connector Sockets | Bulgin | SA3149/1 | |
| Sylgard 184 Silicone Elastomer | Dow Corning | ||
| 100 x 50 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 | |
| Silk Sutures | Ethicon | K89OH | |
| 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 | |
| Yaki Sushi Nori Seaweed | Rhee Bros | ||
| Kimwipes | Kimberly-Clark | 34155 |
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Tags
In Vitro PreparationEliciting Feeding Motor ProgramsRecording Physiological MovementsAplysia CalifornicaMultifunctionalityFeeding ApparatusBuccal MassGangliaIdentified NeuronsElectrophysiological StudyMotor ProgramsIngestive ProgramsEgestive ProgramsIsolated GangliaMuscle MovementsReal BehaviorsNerve RecordingsMuscle RecordingsIndividual NeuronsIn Vivo Recordings
Cite This Article
McManus, J. M., Lu, H., Chiel, H. J. An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica. J. Vis. Exp. (70), e4320, doi:10.3791/4320 (2012).