Preparation and Maintenance of Organotypic Cultures for Multi‐Electrode Array Recordings

Veronica C. Karpiak1, Dietmar Plenz1

1 Laboratory of Systems Neuroscience, NIMH NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.15
DOI:  10.1002/0471142301.ns0615s19
Online Posting Date:  August, 2002
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Abstract

Preparation and Maintenance of Organotypic Cultures for Multi‐Electrode Array Recordings (Veronica C. Karpiak and Dietmar Plenz, National Institutes of Mental Health, NIH, Bethesda, Maryland). Recording from neuronal cultures with multi‐electrode arrays (MEAs) provides a powerful tool for studying neuronal activity with many neurons simultaneously in vitro. This unit describes the detailed steps necessary for growing organotypic cultures on MEAs and the typical neuronal activity that is obtained with this methodology.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • 100% ethanol
  • Chicken plasma (Sigma‐Aldrich)
  • Brain slices (unit 3.3 or unit 6.11)
  • 50 to 175 or 175 to 350 NIH units/mg thrombin (factor IIa) from bovine plasma (Sigma‐Aldrich)
  • MEA medium, 35°C (see recipe)
  • Mitosis inhibitor (see recipe)
  • 0.1% protease solution (e.g., trypsin; Sigma‐Aldrich)
  • Cell culture‐grade H 2O, sterile
  • Sylgard 184 silicone elastomer and curing agent (Dow Corning)
  • Sealed culture chamber ring, custom‐made or commercially available (Multi Channel Systems; also see step )
  • 1‐ml syringe equipped with 20‐G × 1 ½‐in. needle
  • 60°C hot plate
  • Multi‐electrode arrays (Multi Channel Systems; see Internet Resources)
  • Sterile class I or II (recommended) culture hood with easily adjustable window
  • 100 W plasma cleaner (PDC‐32G, Harrick) with argon gas, and vacuum pump (Welch)
  • Gauze, sterile
  • 100‐mm polystyrene petri dishes, sterile
  • Flattened microspatulas with 2 × 5‐mm blade, sharpened with descending grades of abrasive paper and polished on leather
  • Cooled surface (e.g., blue ice blocks, frozen; working range: 0° to 4°C)
  • Rubber O‐ring (11/16‐in. i.d. × 7/8‐in. o.d. × 3/32‐in. wide; Small Parts Inc.)
  • 35°C incubator
  • Sonicator
  • Additional reagents and equipment for cell culture (unit 3.3 or unit 6.11)
NOTE: The MEA electrodes should never be directly touched with any object and outer electrode contacts and gold leads must be protected from scratches and medium spillage.
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Figures

Videos

Literature Cited

Literature Cited
   Droge, M.H., Gross, G.W., Hightower, M.H., and Czisny, L.E. 1986. Multielectrode analysis of coordinated, multisite, rhythmic bursting in cultured CNS monolayer networks. J. Neurosci. 6:1583‐1592.
   Egert, U., Schlosshauer, B., Fennrich, S., Nisch, W., Fejtl, M., Knott, T., Müller, T., and Hämmerle, H. 1998. A novel organotypic long‐term culture of the rat hippocampus on substrate‐integrated multielectrode arrays. Brain Res. Brain Res. Protoc. 2:229‐242.
   Gähwiler, B.H., Capogna, M., Debanne, D., McKinney, R.A., and Thompson, S.M. 1997. Organotypic slice cultures: A technique has come of age. Trends Neurosci. 20:471‐477.
   Gross, G.W., Rhoades, B.K., Reust, D.L., and Schwalm, F.U. 1993. Stimulation of monolayer networks in culture through thin‐film indium‐tin oxide recording electrodes. J. Neurosci. Methods 50:131‐143.
   Hatsopoulos, N.G., Ojakangas, C.L., Paninski, L., and Donoghue, J.P. 1998. Information about movement direction obtained from synchronous activity of motor cortical neurons . Proc. Natl. Acad. Sci. U.S.A. 95:15706‐1571.
   Nicolelis, M.A., Ghazanfar, A.A., Stambaugh, C.R., Oliveira, L.M., Laubach, M., Chapin, J.K., Nelson, R.J., and Kaas, J.H. 1998. Simultaneous encoding of tactile information by three primate cortical areas. Nat. Neurosci. 1:621‐630.
   Nisch, W., Bock, J., Egert, U., Hämmerle, H., and Mohr, A. 1994. A thin film microelectrode array for monitoring extracellular neuronal activity in vitro. Biosens. Bioelectron. 9:737‐741.
   Plenz, D. and Kitai, S.T. 1998. “Up” and “down” states in striatal medium spiny neurons simultaneously recorded with spontaneous activity in fast‐spiking interneurons studied in cortex‐striatum‐substantia nigra organotypic cultures. J. Neurosci. 18:266‐283.
   Plenz, D., Herrera‐Marschitz, M., and Kitai, S.T. 1998. Morphological organization of the globus pallidus‐subthalamic nucleus system studied in organotypic cultures. J. Comp. Neurol. 397:437‐457.
   Stoppini, L., Duport, S., and Correges, P. 1997. A new extracellular multirecording system for electrophysiological studies: Application to hippocampal organotypic cultures. J. Neurosci. Methods 72:23‐33.
   Thiebaud, P., de Rooij, N.F., Koudelka‐Hep, M., and Stoppini, L. 1997. Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures. IEEE Trans. Biomed. Eng. 44:1159‐1163.
   Vaadia, E., Haalman, I., Abeles, M., Bergman, H., Prut, Y., Slovin, H., and Aertsen, A. 1995. Dynamics of neuronal interactions in monkey cortex in relation to behavioural events. Nature 373:515‐518.
Internet Resources
  http://www.multichannelsystems.com
  Website of Multi Channel Systems for further information on MEA purchase.
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