Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)
Summary
This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.
Abstract
The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons. These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4). Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6). For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6). Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly. In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons. The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.
Protocol
1. Preparation for the dissection: Select the appropriate culture dish (10 cm or 6-well or 24-well plates, etc.) depending on the nature of your experiments, and coat them with rat-tail collagen 24 hours before dissection. The methods for preparing rat-tail collagen and using it to coat culture dishes have been described elsewhere (1). Prepare a 0.25% Trypsin solution in phosphate buffered saline (PBS) (no EDTA) and filter sterilize. Prepare 1 ml aliquots and store at -20ºC. Prepare a s…
Discussion
We use Sprague Dawley rats for our cultures.
Take time and care when cleaning the ganglia. Remove all debris, blood vessels and fat. This step is important in ensuring a culture that is more or less homogeneous and free of extraneous cell types. The addition of uridine/5-FDU will largely eliminate any remaining non-neuronal (mitotic) cells contaminating the culture or at least suppress their proliferation.
In addition, during the dissociation step, be patient and…
Disclosures
The authors have nothing to disclose.
Acknowledgements
NZ would like to thank lab members Subhas Biswas, Andrew Sproul, and Ryan Willet for training her in dissecting and harvesting sympathetic neurons. Supported by grants from the NIH-NINDS.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Forceps, Stainless steel #5 | Tool | Roboz | RS4976 | |
| Scissors, DEAVER straight sharp-blunt- 43mm blades – 5.5 | Tool | Roboz | RS6760 | |
| RPMI 1640 w/ L-Glutamin | Reagent | Cellgro, Mediatech, Inc. | 10-040-CV | |
| Fetal Bovine Serum | Reagent | SAFC Biosciences | 12103c | |
| Donor Horse Serum | Reagent | JRH Biosciences | 12449 | Heat-inactivate by incubating in 56°C waterbath for 30 minutes. |
| Penicillin/streptomycin | Reagent | Gibco, Invitrogen | 15140-122 | |
| Trypsin without EDTA | Reagent | Difco | MT 25-050-CI | |
| Uridine | Reagent | Sigma | U3003 | |
| 5-Fluoro-5′ deoxyuridine | Reagent | Sigma | F-8791 | |
| NGF | Reagent | Harlan Bioproduct | BT-5025 | |
| Dissection Microscope and light source | Microscope | |||
| 15 ml polypropylene tubes | Tool | BD Falcon | 352096 | |
| Cell culture dishes | Tool | Corning, Nunclone |
References
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Cite This Article
Zareen, N., Greene, L. A. Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG). J. Vis. Exp. (23), e988, doi:10.3791/988 (2009).