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Culture of Substantia Nigra Neurons

Lorenz Studer¹

¹National 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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Abstract

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 free-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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Unit Introduction
Basic Protocol: 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
Bibliography
Figures

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Materials

Basic Protocol: Preparation of Dissociated Nigral Cell Cultures

Materials

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 3F 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

Additional Materials (also see Basic Protocol)

Neurobasal medium/2% B27 (Neurobasal/B27; see recipe)
Dry 37°C, 5% CO₂ incubator
15-ml (17 × 120–mm) conical-bottom polystyrene tubes (Falcon), stored in 5% CO₂ incubator for at least 24 hr prior to use
Roller drum (Bellco)

Alternate Protocol 2: Preparation of Matrix-Based Organotypic Cultures

Additional Materials (also see Basic Protocol)

5 ml lyophilized chicken plasma (Sigma or Cocalico) reconstituted in 5 ml H₂O
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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Figures

Figure 3.3.1

Schematic illustration of the three culturing procedures described in the text. DIS (dissociated) cultures: The generation of dissociated mesencephalic cultures is carried out by triturating the dissected pieces and plating the cell suspension on precoated petri dishes or chamber slides. FFRT (free-floating rotor tube) cultures: Dissected tissue pieces are further divided either manually or by means of a tissue chopper and then directly added to the culture medium. CP (chicken plasma) cultures: Dissected tissue pieces further divided either manually or by means of a tissue chopper and then embedded in a chicken plasma matrix coagulated by the addition of thrombin. The glass coverslip with the embedded tissue attached is inserted into a culture tube. The tubes of FFRT and CP cultures are then placed in a roller drum.

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Figure 3.3.2

Dissection of fetal ventral mesencephalon. (A) Overview of mesencephalic flexure of E-12 rat embryo. Cuts are made along the indicated lines to isolate the mesencephalic region encompassing the anlage of the substantia nigra. (B) The dorsal mesencephalon (tectum anlage) is slit medially. (C) Same piece as in B now exhibits butterfly-like shape. Lateral parts are cut away along the indicated lines trying to tear meninges to the side. (D) Loosened meninges can be readily recognized (arrows) and are carefully separated from the CNS tissue using a Dumont no. 5 forceps to hold the meninges and a 30.5-G needle used as a microknife. (E) The final size of the dissected piece is ~1 × 1.5 × 0.3 mm. The arrows point to the border between the floor plate and the anlage of the substantia nigra.

View Image
Figure 3.3.3

Cultures of E-12 ventral mesencephalon stained for tyrosine hydroxylase (TH). (A) Dissociated culture grown for 14 days in vitro in DMEM/F-12/N2 supplemented with 10 ng/ml basic fibroblast growth factor (bFGF). TH-immunoreactive cells often appear in dense cell clusters. (B) Cryosection of FFRT culture grown for 7 days in vitro in DMEM/10% FBS. Many immature looking TH-labeled cells are tightly clustered. In other parts of the culture (not shown), the TH-cell density is smaller and individual TH-labeled cells more differentiated. (C) Chicken plasma matrix culture after 9 days in vitro. A substantial degree of flattening allows single cell recognition after this relatively short culture period.

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Literature Cited

Literature Cited
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	Wray, S. 1992. Organotypic slice explant roller-tube cultures. In Practical Cell Culture Techniques (A.A. Boulton, G.B. Baker, and W. Walz, eds.) pp. 201-239. Humana Press, Totowa, N.J..
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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.