隔离的感觉神经元中<em>海兔丫</em谷氨酸能电流膜片钳记录
Summary
我们描述了解剖海洋海兔的神经系统<em>海兔</em麻醉后短期组织培养的神经元，和单细胞离子电流通过膜片钳技术记录的隔离。
Abstract
海洋腹足类软体动物海兔丫有悠长的历史，作为神经系统的功能模型，在学习和记忆研究中具有特别重要的意义。这类研究的典型制剂在其中的感觉和运动神经元保持不变在微创解剖动物，或技术上精心制作的个人感觉和运动神经元的神经细胞共同培养。不太常见的是孤立的小群名义上均匀的神经元是神经制剂，其中分离成单个细胞在短期文化。这种分离的细胞是有用的生物物理特性，使用膜片钳技术，和有针对性的调制这些电导的离子电流。准备这种文化的一种协议。该协议需要容易识别谷氨酸胸膜和颊神经节的感觉神经元中的优势，并介绍了他们的分离和最小的维护，我N培养无血清几天。
Introduction
的海洋opistobranch软体动物， 海兔 ，几十年来一直是一个有用的神经生物学模型。它最出名的习惯和古典空调7，8作为一种模式。在这个模型中对学习和记忆的研究获得了诺贝尔生理学或医学奖的埃里克·坎德尔在2000年，他与阿尔维德·卡尔松和保罗·格林加德10奖品。研究涉及电气录制减少制剂，这种无脊椎动物的神经系统的元素，从动物解剖与神经和肌肉的左连接，这些都有助于阐明海兔的单个神经元的角色。的确切的分子机制，构成海兔学习识别然而，通常采用的另一种技术，长期共培养的感觉神经元和运动神经元，获得从单个供体动物，并允许以形成突触在培养皿21 。
我们和其他1，3，6，14，15，16利用难易程度，确定在这个模型中的神经元可以有针对性的，以及他们的耐力在长期的实验，使游离短期名义上均匀的神经元集群文化我们研究离子电流电压钳下膜片钳配置。许多海兔的神经元站起来，反复多轮膜片钳技术，以便有时间为持久的实验操作。该技术是非常有用的，如神经内分泌袋腹神经节细胞，和我们在这里描述的解离的胸腔和颊神经节的感觉神经元的神经元，但不适合非常大的神经元大于60微米直径的，如L7或R2腹神经节。我们不采用海兔血清在我们的文化，不同的是感觉，运动神经元共培养别处描述。大多数神经元将不使用此程序获得流程为t他第一次在培养的48小时，方便拍摄全细胞电压，但将发芽，约14天死于缺乏的营养物质和/或生长因子的前阐述轴突和其他进程。
该技术生产出每盘50-100个神经元的原代培养的颊和胸膜神经节区域从生理记录。此协议是有用的研究人员在研究单细胞生理学方面的实验，需要无数的实验复制每只动物。这产生一对配对培养由于分离成左，右hemiganglia的靶细胞的解剖，允许匹配的处理的和对照培养物的研究中受益。
该协议的目标颊簇（BSC）的口腔神经节的神经元，并胸膜ventrocaudal（PVC）胸膜神经节神经元。这些细胞是全细胞电压记录和显示ROBUS的适当大小吨谷氨酸回应。讨论的方法是适合大多数在海兔神经系统的神经节。
Protocol
1。细胞的制备在第1天，体重和麻醉动物。 称取30 G-1公斤动物。麻醉1:1海水量在5-10动物：等渗氯化镁。 6H 2 0 1小时，同时通气诸如电动水族馆空气泵连接的气石。 准备夹层用品。 组装干净的夹层托盘用不锈钢直针，如面料销。组装几个培养皿中含有人工海水（ASW 见表1和表3）+青霉素/链霉素（P / S）。 已经准备好一个低?…
Representative Results
在这个协议中的对象内的神经节的感觉神经元的位置，BSC和PVC的神经元是如图1中所示。 BSC的神经元位于口腔神经节的腹侧面向远离的颊质量，完整的神经节（ 图1A），表面上在2个对称的椭圆形集群。 PVC神经元形成双边的，V形的簇，环绕胸神经节的背侧表面向中心轴（ 图1B）。这些感觉神经元的神经节内的刻板位置具有的优点，但缺少的右边或左边的群集?…
Discussion
这里所描述的解离技术产生感觉神经元的文化含有50-100孤立的神经元穿插着小神经胶质细胞和其他不明身份的细胞数。在协议中最重要的步骤是神经节时，保持在酶溶液，轻弹，打散成单个细胞的群集消化的细胞簇的解离。内切酶消化（步骤1.8），必须在可用的温度下进行了优化。在23℃缓慢摇动，13小时是足以消化的的BSC集群while15小时是最佳的PVC集群。在培养皿中的细胞产量低，是由于没有足?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
资助由NIH P40 OD010952，Korein基金会，迈阿密大学奖学金到SLC和ATK美泰奖学金。作者非常感谢工作人员的海兔 ，以及劳伦Simonitis的汉娜·佩克，谁提供了一个数字显微国家资源。
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| Artificial seawater ASW | Sigma-Aldrich | assorted | (mM): 417 NaCl, 10 KCl, 10 CaCl2 (2 H2O), 5MgCl2 (6H2O), 15 HEPES-NaOH, pH 7.6 |
| Intracellular solution | Sigma | assorted | (mM): 450 KCl, 2.9 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4 |
| Poly-D-lysine | Sigma | P6407 | |
| penicillin/streptomycin added to ASW at 1:100 | Lonzo Walkersville, Inc. | 17-603E | 5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin |
| Neutral dispase II | Roche Diagnostics | 10165859001 | |
| hyaluronidase | Sigma-Aldrich | H4272 | |
| collagenase type XI | Sigma-Aldrich | C9407 | |
| L-Glutamate (L-Glu) | Sigma-Aldrich | 49601-100G | |
| D-Aspartate (D-Asp) | Sigma-Aldrich | 11200-10G | |
| N-methyl-D-aspartate (NMDA) | Biomol | 100002-268 | |
| L-Asp | Sigma | A6683-25G | |
| alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) | Sigma | A6816-5MG | |
| L-Glu R antagonists | various | various | |
| agar | |||
| kynurenate | Sigma-Aldrich | 61250 | |
| APV | Sigma-Aldrich | A5282 | |
| DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist) | |||
| 2-propanol | VWRSP | BDH1133 | |
| Chloriding solution | Sigma | assorted | 25 g FeCl3 + 25 ml concentrated HCl + 50 ml H2O |
| Sylgard silicone 2-part polymer | World Precision Instruments (WPI) | SYL184 | Provides pin-out surface for small dissection dishes |
| 0-40x zoom magnification microscope for dissections | Wild | ||
| Techniquip 150 Watts Fiber Optic Illuminator | Microoptics of Florida | TQ FOI-150 | |
| RotoMix 50800 orbital mixer | |||
| Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) & 40x objectives | SR Research Ltd. | Eyelink II | |
| Tektronix digital oscilloscope | SR Research Ltd. | ||
| pClamp 10 data acquisition and analysis software | Molecular Devices | ||
| PC with Windows XP or higher operating system | PC Solutions | Thinkserver with solid state hard drives (80GB) and low noise monitors | |
| Flaming/Brown P87 micropipette puller | Sutter Instruments, Novato, CA | ||
| Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier | Molecular Devices, Sunnyvale, CA | ||
| Axon instruments electrode holder assembly for Axopatch 200B preamplifier | Molecular Devices, Sunnyvale, CA | CV203BU | |
| Digidata 1200 A/D converter | Molecular Devices, Sunnyvale, CA | ||
| Picospritzer, powered by N2 adjustable for pressure and duration | Parker Hannifin, Cleveland, OH | ||
| TMC Micro-G Vibration isolation table | Ametek | ||
| Faraday cage | custom manufacture | ||
| Burleigh Piezoelectric Clamshell Micromanipulators | Burleigh Instruments; Thorlabs | presently PCS-5000; -6000 series + mounts | |
| Narishige M-152 manual manipulators (for perfusion system and picospritzer) | Narishige USA | ||
| Filament pipette glass,1.5 mm OD, 0.84 mm ID – | WPI | 1B150-3 | |
| 3 inch length | |||
| Ag/AgCl half cell | WPI | EP4 | |
| 15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes | VWRSP | 21008-918 | |
| Angled Scissors | Fine Science Tools | 15006-09 | |
| Dumostar Fine forceps | Fine Science Tools | 11295-00 | |
| 35 mm falcon tissue culture dishes | VWRSP | 25382-064 | |
| falcon 150 x 25 mm tissue culture dishes; 1013 | VWRSP | 1013 | also can be made into small dissection dishes with sylgard |
| sylgard | WPI | SYL184 | |
| animal dissection tray | various | ||
| 15 ml centrifuge tubes, 35-2097 BD Falcon | VWRSP | 21008-918 | For 6-bore gravity-fed perfusion system |
| Aluminum clips with screw hole ends | hardware store | For perfusion system | |
| 23 gauge needles (manually file off points) | VWRSP | For perfusion system | |
| Polyethylene tubing 0.022″ID x 0.042″OD; 427411 | Becton-Dickinson | For perfusion system | |
| H-7 pipette stand/holder for microcap perfusion array | Narishige USA | For perfusion system | |
| one-way valves | For perfusion system | ||
| Drummond Microcaps 1 μl | VWRSP | For perfusion system | |
| 18 gauge needles for suction (filed off points) | |||
| Polyethylene tubing | Cole Parmer | 4.27436E+11 | |
| fine dissection pins | Fine Science Tools | 26002-20 | |
| capillary tubes | Kimble 71900-100 | fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS | |
| modeling clay | craft store | ||
| dish holder for microscope stage with isolated ground bath | Custom manufacture | ||
| pasteur pipettes | VWRSP | 14672-412 | |
| pipette bulbs | VWRSP | 53283-911 | |
| acrodisk syringe filters | VWRSP | 28144-040 | |
| thick-walled 1.5 mm diameter borosilicate filament glass | WPI | 1B150F-3 | |
| High purity nitrogen cylinder and bifurcating regulator |
Tables 1-3. Lists of Reagents, Materials, and Equipment.
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Cite This Article
Fieber, L. A., Carlson, S. L., Kempsell, A. T., Greer, J. B., Schmale, M. C. Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents. J. Vis. Exp. (77), e50543, doi:10.3791/50543 (2013).