Acute Isolation of Neurons from the Mature Mammalian Central Nervous System

Alan R. Kay1, David J. Krupa1

1 University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.5
DOI:  10.1002/0471142301.ns0605s00
Online Posting Date:  May, 2001
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Abstract

The acute dissociation procedure provides a simple means of isolating neurons from the mature mammalian central nervous system. The method was primarily devised to isolate neurons for patch‐clamp electrophysiology. It may also prove useful for single‐cell PCR, immunocytochemistry, sorting of fluorescently labeled cells, or long‐term tissue culture of mature neurons. Dissociation is brought about by a combination of proteolysis and an ionic environment that encourages breakdown of the tissue. The method allows the isolation of neurons free of glial ensheathments in as little as 45 min after the sacrifice of the animal. Neurons so isolated lose fine dendritic branches, although the structure proximal to the cell body is often maintained, allowing identification of the morphological type of the neuron. The preparation has the following advantages: (1) the neurons are fully differentiated; (2) the cells are electronically compact, which improves the fidelity of the voltage clamp; (3) the cells are removed from the influence of surrounding cells; and (4) neurons can be isolated from small, circumscribed loci within the adult central nervous system.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • 100% ethanol
  • PIPES saline (see recipe)
  • 100‐ to 300‐g rat
  • Proteinase K solution (see recipe)
  • Trypsin solution (see recipe)
  • Tissue chopper (e.g., Stoelting, McIlwain from Brinkman) or slicer
  • 25‐ml spinner flask (Bellco)
  • 100% oxygen tank and regulator
  • 60‐mm glass petri dish cover
  • Guillotine (e.g., Braintree Scientific)
  • ∼14‐cm scissors
  • 14‐cm fine‐tipped rongeurs
  • Fine‐tipped forceps
  • Spatula with tapered end
  • Size 0 sable‐hair paint brush
  • Scalpel handle and no. 22 blade
  • Pasteur pipet cut to have a wide mouth
  • Magnetic stirrer (e.g., Cole Parmer)
  • Heating box fabricated from aquarium heater, gang valve, and polyethylene (e.g., Tupperware) box (see Fig. )
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Figures

Videos

Literature Cited

Literature Cited
   Aghajanian, G.K. and Rasmussen, K. 1989. Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices. Synapse 3:331‐338.
   Akaike, N., Kaneda, M., Hori, N., and Krishtal, O.A. 1988. Blockade of N‐methyl‐D‐aspartate response in enzyme‐treated rat hippocampal neurons. Neurosci. Lett. 87:75‐79.
   Allen, C.N., Brady, R., Swann, J., Hori, N., and Carpenter, D.O. 1988. N‐methyl‐D‐aspartate (NMDA) receptors are inactivated by trypsin. Brain. Res. 458:147‐150.
   Alonso, A., White, J.A., Oliver, A., and Kay, A.R. 1993. A survey of persistent Na+ currents in rat and human neurons. Soc. Neurosci. Abs. 23:152.
   Blanton, M.G., Lo Turco, J.J., and Kriegstein, A.R. 1989. Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex. J. Neurosci. Methods 30:203‐210.
   Brewer, G.J., Torricelli, J.R., Evege, E.K., and Price, P.J. 1993. Optimized survival of hippocampal neurons in B27‐supplemented Neurobasal, a new serum‐free medium combination. J. Neurosci. Res. 35:567‐576.
   Budde, T., White, J., and Kay, A. 1994. Hyperpolarization‐activated Na+–K+ current (Ih) in neocortical neurons blocked by external proteolysis and internal TEA. J. Neurophysiol. 72:2737‐2742.
   Drewe, J.A., Childs, G.V., and Kunze, D.L. 1988. Synaptic transmission between dissociated adult mammalian neurons and attached synaptic boutons. Science 241:1810‐1813.
   Edwards, F.A., Konnerth, A., Sakmann, B., and Takahashi, T. 1989. A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pfluegers Arch. Eur. J. Physiol. 414:414‐612.
   Ferster, D. and Jagadeesh, B. 1992. EPSP‐IPSP interactions in cat visual cortex studied with in vivo whole‐cell patch recording. J. Neurosci. 12:1262‐1274.
   Kay, A.R. and Connor, J.A. 1990. Preservation of the NMDA response of neurons acutely dissociated from the mature guinea pig hippocampus. J. Neurosci. Methods 33:77‐79.
   Kay, A.R. and Wong, R.K.S. 1986. Isolation of neurons suitable for patch‐clamping from the adult mammalian central nervous systems. J. Neurosci. Methods 16:227‐238.
   Miller, M.W. 1988. Development of projection and local circuit neurons in neocortex. In Cerebral Cortex (A. Peters and E.G. Jones, eds.) pp. 133‐175. Plenum, New York.
   Paulsen, R.E., Contestabile, A., Villani, L., and Fonnum, F. 1987. An in vivo model for studying function of brain tissue temporarily devoid of glial cell metabolism: The use of fluorocitrate. J.Neurochem. 48:1377‐1385.
   Ruoslahti, E. and Pierschbacher, M.D. 1987. New perspectives in cell adhesion: RGD and integrins. Science 238:491‐497.
   White, J.A., Alonso, A., and Kay, A.R. 1993. A heart‐like Na+ current in the medial entorhinal cortex. Neuron 11:1037‐1047.
   White, J.A., Sekar, N.S., and Kay, A.R. 1995. Errors in persistent inward currents generated by space‐clamp errors: A modeling study. J. Neurophysiol. 73:2369‐2377.
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