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Summary
Abstract
Protocol
Discussion
Divulgations
Acknowledgements
Materials
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Neurosciences

从大脑皮层的器官型切片制备全细胞记录

Published: June 03, 2011
doi:
10.3791/2600
, , ,
1Department of Anatomy and Neurobiology,University of Tennessee Health Science Center

Summary

这是一个协议，准备和保持器官的文化一个新皮层锥体神经元的电气录音的目的的片准备。

Abstract

我们一直在研究大鼠大脑皮层锥体神经元电压门控钾通道的表达和功能的作用。由于缺乏这些渠道的具体药理代理商，我们已经采取了遗传学的方法操纵的渠道表达。我们使用一个器官的文化准备（16），以维持细胞的形态和皮质层模式。我们通常会在产后8-10天隔离急性新皮层切片，并保持3-7天文化片。这使我们能够在我们与急性切片的工作的一个年龄相仿的研究神经元和最大限度地减少了旺盛切片中的兴奋连接的发展。我们从视觉识别的锥体神经元层的II / III或V使用红外线照射（红外）和微分干涉对比显微镜（DIC）在电流或电压钳全细胞膜片钳记录。我们使用基因枪的野生型或突变的钾离子通道的DNA（基因枪）转来操纵表达的渠道，研究其功能。 epifluorescence显微镜与绿色荧光蛋白（GFP）基因共转染后的转染细胞很容易识别。我们比较对相邻的未转染的神经元，在同一层的同一片转染细胞的录音。

Protocol

1。一天的切片前的准备工作我们发现这是更有效的反应釜的手术器械和准备的解决方案前一天的切片。 高压灭菌仪器。 （半无菌条件下进行的手术和切片）。高压灭菌器下面的包，分别在高压灭菌纸包裹： 手术包：铲，22号手术刀刀片手柄，剪刀，镊子 切片包装：3旋钮（刀片控股旋钮和2浴，确保旋钮）和刀片护罩?…

Discussion

我们一直在研究在大鼠大脑皮层（4 9-11）锥体神经元电压门控钾通道的表达和功能的作用。由于缺乏这些渠道的具体药理代理商，我们使用一种遗传方法操纵通道表达（1,14,15,17-19）。我们利用一个器官的文化准备（2,3; 5-8; 12,13,15-22）。Stoppini等（16）的方法修改，以维持细胞的形态和皮质层模式。我们在产后6-17天隔离急性新皮层切片，并保持2-7天文化片。这使得我们研究类似岁的神经元在?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者想感谢真弓樱和丽贝卡Foehring优秀的技术援助。此外，我们要感谢博士。罗德里戈安德拉德在实施为我们提供了转基因构造的器官切片文化和基因枪转染协议的博士和珍妮Nerbonne的援助。这项工作是由美国国立卫生研究院授予：NS044163支持NINDS（RCF）。

Materials

Surgery / transfection / culture:

  1. Brain Slicer: Campden Vibroslice #MA572 World Precision Instruments, Sarasota, FL, USA
  2. Gene Gun System: Bio-Rad Helios # 165-2431 (Bio-Rad Laboratories, 1000 Alfred Nobel Drive, Hercules, CA 94547)
    • Includes: Gene gun, helium hose assembly with regulator, tubing prep station (#165-2418), syringe kit, Tefzel tubing, tubing cutter, optimization kit (#165-2424), tubing cutter
    • Bio-Rad Helium Regulator (#165-2413)
    • disposable supplies for Helios from Bio-Rad:
      • 1.6 μm Gold Microcarriers: #165-2264
      • Tefzel Tubing: #165-2441
  3. Incubator: Forma Scientific model # 3110 (Thermo-Scientific: (866) 984- 3766).

Media:

  1. Horse Serum: Hyclone donor equine #SH 30074. (HyClone, 925 West 1800 South, Logan, UT 84321)
  2. HMEM (Minimal Essential Media plus HBSS and HEPES, no glutamine: Lonza BioWhittaker Catalog #12-137F): GIBCO/INVITROGEN, (800) 955- 6288, Option 1.
  3. HBSS (GIBCO Hanks buffered saline, #24020-117): GIBCO/INVITROGEN, (800) 955- 6288, Option 1.
  4. MEM (GIBCO minimal essential medium, #12360-038), GIBCO/INVITROGEN, (800) 955- 6288, Option 1.
  5. 250 mL Millipore 0.2 μm filter: #SC6PU02RE
  6. Plastic Transfer pipettes: Fisher #13-711-20.
  7. 50 mL Millipore steriflip 0.22 μm filter (#SCGP00525)

Items 6-8 obtained from: Fisher Scientific, 1241 Ambassador Blvd, P.O. Box 14989, St. Louis, MO 63132.

Recording:

  1. Pipet glass: Harvard GC150TF-10: Harvard Apparatus, 84 October Hill Road, Holliston, Massachusetts 01746
  2. Sutter P-87 horizontal electrode puller: Sutter Instrument Company, One Digital Drive, Novato, CA 94949
  3. Axon Instruments Multiclamp 700B amplifier: Molecular Devices, Inc. 1311 Orleans Drive, Sunnyvale, CA 94089-1136
  4. PClamp 10 data acquisition software: Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089-1136
  5. lectrode position is controlled with Sutter ROE-200 manipulators and PC-200 controller: Sutter Instrument Company, One Digital Drive, Novato, CA 94949.
  6. Microscope: Olympus BX-50WI upright microscope with IR-DIC optics
  7. IR-sensitive camera OLY-150 (Olympus) or DAGE-MTI (DAGE-MTI, 01 North Roeske Avenue, Michigan City, IN 46360).
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References

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  2. Buonomano, D. V. Timing of neural responses in cortical organotypic slices. Proc Natl Acad Sci U S A. 100, 4897-4902 (2003).
  3. Caeser, M., Bonhoeffer, T., Bolz, J. Cellular organization and development of slice cultures from rat visual cortex. Exp Brain Res. 77, 234-244 (1989).
  4. Foehring, R. C., Toleman, T., Higgs, M., Guan, D., Spain, W. J. Actions of Kv2.1 channels in rat neocortical pyramidal neurons. Soc Neurosci Abstr. 34, (2009).
  5. Gähwiler, B. H. Organotypic monolayer cultures of nervous tissue. J Neurosci Methods. 4, 329-342 (1981).
  6. Gähwiler, B. H. Organotypic cultures of neural tissue. Trends Neurosci. 11, 484-489 (1988).
  7. Gähwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A., Thompson, S. M. Organotypic slice cultures: a technique has come of age. Trends Neurosci. 20, 471-477 (1997).
  8. Gähwiler, B. H., Thompson, S. M., Muller, D. Preparation and Maintenance of Organotypic Slice Cultures of CNS Tissue. Current Protocols in Neuroscience. , 6.11.1-6.11.11 (2001).
  9. Guan, D., Lee, J. C., Tkatch, T., Surmeier, D. J., Armstrong, W. E., Foehring, R. C. Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones. J Physiol. 571, 371-389 (2006).
  10. Guan, D., Lee, J. C. F., Higgs, M., Spain, W. J., Armstrong, W. E., Foehring, R. C. Functional roles of Kv1 containing channels in neocortical pyramidal neurons. J. Neurophysiol. 97, 1931-1940 (2007).
  11. Guan, D., Tkatch, T., Surmeier, D. J., Armstrong, W. E., Foehring, R. C. Kv2 subunits underlie slowly inactivating potassium current in rat neocortical pyramidal neurons. J Physiol. 581, 941-960 (2007).
  12. Johnson, H. A., Buonomano, D. V. A method for chronic stimulation of cortical organotypic cultures using implanted electrodes. Neurosci Methods. 176, 136-143 (2009).
  13. Johnson, H. A., Buonomano, D. V. Development and plasticity of spontaneous activity and Up states in cortical organotypic slices. J Neurosci. 27, 5915-5925 (2007).
  14. Malin, S. A., Nerbonne, J. M. Delayed rectifier K+ currents, IK, are encoded by Kv2 alpha-subunits and regulate tonic firing in mammalian sympathetic neurons. J Neurosci. 22, 10094-10105 (2002).
  15. O’Brien, J. A., Holt, M., Whiteside, G., Lummis, S. C., Hastings, M. H. Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue. J Neurosci Methods. 112, 57-64 (2001).
  16. Stoppini, L., Buchs, P. A., Muller, D. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods. 37, 173-182 (1991).
  17. Villalobos, C., Shakkottai, V. G., Chandy, K. G., Michelhaugh, S. K., Andrade, R. SKCa channels mediate the medium but not the slow calcium-activated afterhyperpolarization in cortical neurons. J Neurosci. 24, 3537-3542 (2004).
  18. Walker, P. D., Andrade, R., Quinn, J. P., Bannon, M. J. Real-time analysis of preprotachykinin promoter activity in single cortical neurons. J Neurochem. 75, 882-885 (2000).
  19. Woods, G., Zito, K. Preparation of gene gun bullets and biolistic transfection of neurons in slice culture. J Vis Exp. , (2008).
  20. O’Brien, J. A., Lummis, S. C. Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat Proc. 1, 977-981 (2006).
  21. Joshi, P., Dunaevsky, A. Gene gun transfection of hippocampal neurons. J Vis Exp. , (2006).
  22. Biewanga, J. E., Destree, O. H., Scharma, L. H. . J Neurosci Met. 71, 67-75 (1997).

Tags

Whole Cell RecordingOrganotypic Slice PreparationNeocortexVoltage-gated Potassium ChannelsPyramidal NeuronsGenetic ApproachOrganotypic Culture PreparationLaminar Pattern Of CortexAcute Neocortical SlicesVisually-identified Pyramidal NeuronsInfrared IlluminationDifferential Interference Contrast MicroscopyWhole Cell Patch ClampCurrent-clampVoltage-clampBiolistic TransfectionWild Type Potassium Channel DNAMutant Potassium Channel DNAFunction StudyEpifluorescence MicroscopyGreen Fluorescent Protein (GFP)Adjacent Untransfected Neurons
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Foehring, R. C., Guan, D., Toleman, T., Cantrell, A. R. Whole Cell Recording from an Organotypic Slice Preparation of Neocortex. J. Vis. Exp. (52), e2600, doi:10.3791/2600 (2011).
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