Culture of Substantia Nigra Neurons

Lorenz Studer1

1 National Institute of Neurological Disorders and Stroke, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.3
DOI:  10.1002/0471142301.ns0303s00
Online Posting Date:  May, 2001
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Primary cultures of nigral tissue are widely used as a model system to assay effects of trophic and toxic agents on dopaminergic neurons. Cultured dopaminergic neurons have been successfully transplanted in animals and led to behavioral improvement in animal models of Parkinson's disease. Cell cultures have also been used to study the development of substantia nigra, allowing investigators to identify early inductive events important for nigral development and to study dopaminergic differentiation and target innervation. This unit provides simple and reliable culture protocols for these applications. The first approach presented is the preparation of dissociated nigral cell cultures, the later steps of which can be used as a simple and efficient assay for testing growth factors. A second approach is the preparation of freeā€floating roller tube cultures, which may be used as a tool for neural transplantation and to study more complex developmental events. A third approach is the production of organotypic cultures using chicken plasma as a matrix. Organotypic cultures can maintain the in vivo cytoarchitecture of a host region in vitro.

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Table of Contents

  • Basic Protocol 1: Preparation of Dissociated Nigral Cell Cultures
  • Alternate Protocol 1: Preparation of Free‐Floating Roller Tube (FFRT) Cultures
  • Alternate Protocol 2: Preparation of Matrix‐Based Organotypic Cultures
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Preparation of Dissociated Nigral Cell Cultures

  • Pregnant Sprague‐Dawley rat
  • Dissection medium (see recipe), ice cold
  • DMEM/F‐12/N2 medium ( appendix 2A)
  • 0.4% trypan blue, or (if inverted fluorescence microscope is available) two‐color fluorescence cell viability assay (Molecular Probes)
  • Dissection tools:
    • Small and medium‐sized scissors
    • 2 small spatulas
    • 2 sets of Dumont no. 5 forceps
    • 30.5‐G needles (Thomas) or sharpened tungsten needles, attached to 1‐ml Luer syringes, to be used as sterile microknives
    • Dissecting microscope
  • 10‐cm polystyrene tissue culture dishes (Falcon)
  • 5‐ml glass pipet (Falcon)
  • 15‐ml (17 × 120–mm) conical‐bottom polystyrene tubes (Falcon)
  • 6‐ or 10‐cm tissue culture dishes or 8‐well glass chamber slides, coated with poly‐D‐lysine ( appendix 2A)
  • Additional reagents and equipment for counting cells with a hemacytometer and trypan blue (see CPMB APPENDIX and appendix 1A in this manual)
NOTE: Autoclave all instruments before use, and clean them as needed during the dissection procedure in 100% ethanol and then in sterile dissection medium. During dissection, store all embryonic tissue in dissection medium on ice.

Alternate Protocol 1: Preparation of Free‐Floating Roller Tube (FFRT) Cultures

  • Neurobasal medium/2% B27 (Neurobasal/B27; see recipe)
  • Dry 37°C, 5% CO 2 incubator
  • 15‐ml (17 × 120–mm) conical‐bottom polystyrene tubes (Falcon), stored in 5% CO 2 incubator for at least 24 hr prior to use
  • Roller drum (Bellco)

Alternate Protocol 2: Preparation of Matrix‐Based Organotypic Cultures

  • 5 ml lyophilized chicken plasma (Sigma or Cocalico) reconstituted in 5 ml H 2O
  • 200 U/ml thrombin (see recipe)
  • Supplemented DMEM/HBSS/10% FBS (see recipe)
  • Standard tissue chopper (McIlwain from Brinkman)
  • 12 × 24–mm glass coverslips, cleaned and sterilized (see recipe)
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Literature Cited

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Key References
   Takeshima et al., 1996. See above.
  Presents a method of dissociated mesencephalic culture designed for standardizing purposes that is in many ways congruent with the type of dissociated culture described in this unit.
   Spenger et al., 1994. See above.
  Methodological paper giving the first description of the free‐floating roller tube culture system.
   Gühwiler, 1981. See above.
  Methodological paper giving the detailed protocol for organotypic chicken plasma cultures of various CNS regions, of which the protocol in this unit is a modified version.
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