Name: Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG) (Video) | JoVE Journal
Uploaded: 2009-01-30T00:00:00.000+00:00
Duration: 11 min 49 s
Description: Learn to isolate and culture primary sympathetic neurons from rat superior cervical ganglia using enzymatic dissociation and collagen-coated plates. This protocol enables neurobiological studies of autonomic nervous system function in vitro.

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5 minutes

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.

1.	Preparation for the dissection:
<ol>
<li>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).</li>
<li>Prepare a 0.25% Trypsin solution in phosphate buffered saline (PBS) (no EDTA) and filter sterilize. Prepare 1 ml aliquots and store at -20&ordm;C.</li>
<li>Prepare a solution containing 2.5 mM each of Uridine and 5-FDU in distilled/deionized H2O. Filter-sterilize and prepare 50 &micro;l aliquots. Store at -20&ordm;C.</li>
<li>Prepare Complete Medium: RPMI 1640 culture medium with 10% heat-inactivated horse serum (heat inactivate by incubating in a 56&ordm;C waterbath for 30 minutes) and 5% fetal bovine serum.</li>
<li>Prepare Final Medium: RPMI 1640 culture medium with 1% heat-inactivated horse serum + NGF (100&eta;g/ml final concentration) + penicillin/streptomycin (1/250) + 1/250 uridine/5-FDU solution (10 &micro;M final concentration). Note: this medium should be made up fresh before use every time. For an experiment in a 24-well plate format, one will need to prepare 12 ml of this medium. It is recommended that you prepare a little extra. NGF can be purchased from a variety of commercial sources (see Table at the end of this protocol).</li>
<li>Equipment: One will need a dissecting microscope and light source. A dual gooseneck fiber optic illuminator with focusing tips is preferred. In addition, one will need to clean and sterilize (by autoclaving or soaking in 70% ethanol for at least 20 minutes) the following dissection tools: two pairs of micro-dissecting stainless steel forceps, and a pair of dissecting scissors (also stainless steel and both from Roboz). Two sterile syringe needles (26 gague x 1 &frac12; inches or similar) will also be needed. It is necessary to set up a dissection board, which is a 3&rdquo;x3&rdquo; piece of styrofoam wrapped in aluminum foil and covered with a Kimwipe. Spray the board with 70% ethanol to decontaminate. Finally, prepare a fire-polished glass pasture pipette. In order to fire-polish, take the glass pipette and hold the tip in the flame for a few seconds, while rotating. Upon inspection, the tip will look smoother and the opening will be narrower. This is necessary for the step where the cells will be dissociated. Perform fire-polishing in a cell culture hood so that the pipette remains sterile.</li>
</ol>
2. Dissection of Superior Cervical Ganglia:
<ol>
<li>Collect neonatal rat pups that are of age P0 or P1.</li>
<li>Rapidly wipe the pup for dissection with 70% ethanol to reduce chances of contamination.</li>
<li>Rapidly decapitate pup with a sharp pair of scissors (this step will not be shown in the video). Hold the head using sterile forceps against sterile gauze to briefly drain excess blood.</li>
<li>Place the head on the dissection pad with the ventral side facing up and the caudal end oriented toward you. The severed trachea should be readily visible. Use the sterile syringe needles to secure the severed head to the dissecting pad in the following manner: push one pin though the roof of the mouth into the pad and push the second pin though the severed trachea into the pad. Place the pad under the dissecting microscope and begin dissection.</li>
<li>Using both forceps (one in each hand) clear away skin and fat around the trachea, taking care not to go too deep. Doing this will expose a pair of muscles running almost parallel to each other on either side of the trachea. Note: during dissection, you will need to irrigate with cold sterile PBS or cold, serum free RPMI 1640 medium delivered with a sterile, Pasteur pipette to wash away excess blood and to keep the area clean and moist. This may be done only once or more than once depending on how fast one works. For an experience dissector, it takes only about two to three minutes to dissect out a pair of ganglia.</li>
<li>Use the forceps to cut and remove the muscle on one side first.</li>
<li>Once the muscle is removed, the carotid artery will become visible. Often it is light pinkish and contains blood. It may appear lighter in color than other blood vessels that you may see in the vicinity and for this reason the carotid artery may not be noticeable right away. This artery runs alongside the trachea and bifurcates into what looks like a &ldquo;Y&rdquo;-shaped ribbon. This bifurcation is an important landmark and once it is located, it will be relatively easy to find the SCG.</li>
<li>Sever the artery at the most caudal end and lift it up slightly with the forceps (the other end is still attached). Once you do this, you will see an almond-shaped, glossy looking small mass of tissue that is loosely attached to the artery just at the point of bifurcation and that has thread-like pre- and/or post-ganglionic connectives. This is the superior cervical ganglion. Use a second pair of forceps in the other hand to gently detach it and place in a small culture dish (35 mm) containing cold, serum free RPMI 1640 medium.</li>
<li>Next, extract the ganglion on the other side of the trachea following the same protocol. </li>
</ol>
3.	Cleaning the Ganglia:
<ol>
<li>Once all the ganglia have been dissected out, they should be freed as much as possible of extraneous tissue.</li>
<li>Under the dissection microscope, you may see that the ganglia have attached pieces of carotid artery, fat or other tissue. Work with forceps in each hand to tease away these unwanted materials. Place cleaned ganglia into a sterile 15ml conical, polypropylene tube, containing RPMI 1640 medium.</li>
<li>The pre- and/or post-ganglionic connectives should also be trimmed away. Each ganglion has about 10,000 neurons. Once the neurons are plated, they will regrow their neurites.</li>
</ol>
4. Establishing Sympathetic Neuron culture:
<ol>
<li>Centrifuge the ganglia at 200xg for 2 minutes and discard the supernatant.</li>
<li>Resuspend the ganglia in 0.5 to 1 ml of 0.25% trypsin solution. Place in a 37&#x02DA;C waterbath or incubator for 30 minutes. This will help dissociate the cells in the ganglia.</li>
<li>After 30 minutes, add 10 ml of Complete Medium to dilute out and neutralize the trypsin and centrifuge at 200xg for 2 minutes.</li>
<li>Discard supernatant and resuspend in 2 ml of Final Medium.</li>
<li>Further dissociate the cells by triturating the ganglia up and down with the fire-polished glass pasture pipette. Triturate the ganglia 20 times and place the tube on ice for a few minutes.</li>
<li>Meanwhile, fire-polish the pipette more to make the opening narrower (approximately 2/3 to 1/2 mm in diameter). Wait a few minutes until it cools and triturate the ganglia 20 times more. As you continue, the medium will become progressively cloudier indicating that the cells are being dissociated. Repeat this step once or twice more depending on how well the cells dissociate. After some time, you will notice that all the ganglia have dissociated into neurons and no intact ganglia are visible in the tube. There may be some tissue material that will not dissociate. At this point, let the tube sit in ice for a few minutes so that any undissociated debris can settle to the bottom. Now carefully collect almost all of the medium from the top and place in another tube. To ensure that you do not collect the undissociated debris, leave about 50 &micro;l at the bottom of the tube. Add more of the Final medium to the tube containing dissociated cells: this volume depends on the type of culture plate one is using to culture the neurons. For example: if a 24-well dish is to be employed, then plate 0.5 ganglia per well (~10,000 neurons per well) in 0.5 ml of Final medium per well. A typical rat litter consists of 12 pups, which would give 24 ganglia. This is enough to seed one 24-well plate and the total volume of Final medium is 12 ml. Note: the ganglia also contain a small population of glia and other cell types that will be present in the culture. Using Uridine/5-FDU in the Final medium will prevent their proliferation and promote their death.</li>
<li>Feed cells every 48 hours by replacing 2/3 of the medium with fresh RPMI +1% horse serum + NGF and uridine/5-FDU. Note: At first, the culture contains sympathetic neurons that look round with no neurites. Short neurites become visible 24 hours after dissection and progressively become denser and more branched over time.</li>
</ol>

<ol><li>Banker, G., Goslin, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBanker%2C+G+%22Culturing+Nerve+Cells%22">Culturing Nerve Cells</a>. , MIT Press. (1998).</li>
<li>Biswas, S. C., Shi, Y., Sproul, A., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pro-apoptotic+Bim+Induction+in+Response+to+Nerve+Growth+Factor+Deprivation+Requires+Simultaneous+Activation+of+Three+Different+Death+Signaling+Pathways%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways.</a> The Journal of Biological Chemistry. 282 (40), 29368-29374 (2007).</li>
<li>Bocchini, V., Angeletti, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Nerve+Growth+Factor%3A+purification+as+a+30%2C000-molecular-weight+protein%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">The Nerve Growth Factor: purification as a 30,000-molecular-weight protein.</a> Proc. Natl. Acad. Sci. USA. (64), 787-794 (1969).</li>
<li>Dechant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chat+in+the+Trophic+Web%3A+NGF+Activates+Ret+by+Inter-RTK+Signaling%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Chat in the Trophic Web: NGF Activates Ret by Inter-RTK Signaling.</a> Neuron. 33 (2), 156-158 (2002).</li>
<li>Deckwerth, T., Elliott, J. L., Knudson, C. M., Johnson, E. M., Snider, W. D., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bax+is+required+for+neuronal+death+after+trophic+factor+deprivation+and+during+development%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Bax is required for neuronal death after trophic factor deprivation and during development.</a> Neuron. (17), 401-411 (1996).</li>
<li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+in+Vitro+Studies+on+the+Nerve+Growth+Factor+%28NGF%29+Requirement+of+Neurons+-+I.+Sympathetic+Neurons%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Quantitative in Vitro Studies on the Nerve Growth Factor (NGF) Requirement of Neurons - I. Sympathetic Neurons.</a> Developmental Biology. (58), 96-105 (1977).</li>
<li>Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cyclin-dependent+kinases+participate+in+death+of+neurons+evoked+by+DNA-damaging+agents%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.</a> The Journal of Cell Biology. 143, No 2 457-467 (1998).</li>
<li>Pierchala, B. A., Ahrens, R. C., Paden, A. J., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nerve+Growth+Factor+Promotes+the+Survival+of+Sympathetic+Neurons+through+the+Cooperative+Function+of+the+Protein+Kinase+C+and+Phosphatidylinositol+3-Kinase+Pathways%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Nerve Growth Factor Promotes the Survival of Sympathetic Neurons through the Cooperative Function of the Protein Kinase C and Phosphatidylinositol 3-Kinase Pathways.</a> The Journal of Cell Biology. 279, No 27 27986-27993 (2004).</li>
</ol>

The authors have nothing to disclose.

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...

<table><tbody><tr><td>Forceps, Stainless steel #5</td><td>Tool</td><td>Roboz Surgical Instruments Co.</td><td>RS4976</td><td></td></tr><tr><td>Scissors, DEAVER straight sharp-blunt- 43mm blades - 5.5</td><td>Tool</td><td>Roboz Surgical Instruments Co.</td><td>RS6760</td><td></td></tr><tr><td>RPMI 1640 w/ L-Glutamin</td><td>Reagent</td><td>Cellgro</td><td>10-040-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Reagent</td><td>SAFC Global</td><td>12103c</td><td></td></tr><tr><td>Donor Horse Serum</td><td>Reagent</td><td>JRH Biosciences</td><td>12449</td><td>Heat-inactivate by incubating in 56&amp;#x00B0;C waterbath for 30 minutes.</td></tr><tr><td>Penicillin/streptomycin</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Trypsin without EDTA</td><td>Reagent</td><td>Difco Laboratories</td><td>MT 25-050-CI</td><td></td></tr><tr><td>Uridine</td><td>Reagent</td><td>Sigma-Aldrich</td><td>U3003</td><td></td></tr><tr><td>5-Fluoro-5&amp;rsquo; deoxyuridine</td><td>Reagent</td><td>Sigma-Aldrich</td><td>F-8791</td><td></td></tr><tr><td>NGF</td><td>Reagent</td><td>Harlan Laboratories</td><td>BT-5025</td><td></td></tr><tr><td>Dissection Microscope and light source</td><td>Microscope</td><td></td><td></td><td></td></tr><tr><td>15 ml polypropylene tubes</td><td>Tool</td><td>BD Biosciences</td><td>352096</td><td></td></tr><tr><td>Cell culture dishes</td><td>Tool</td><td>Corning</td><td></td><td></td></tr></tbody></table>

protocol for culturing sympathetic neurons from rat superior cervical ganglia (scg)

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

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.

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

This procedure begins with isolating the superior cervical sympathetic ganglia from P zero neonatal rats. Each brain has two superior cervical sympathetic ganglia. Approximately 24 ganglia can be harvested from a single litter of rat pups.The dissociation of the ganglia begins with trips and treatment for 30 minutes. Further dissociation is accomplished via repeated reation with a fire polished pester pipette resulting in a single cell suspension. Sympathetic neurons are then plated on collagen ENC coated dishes.Hi, I am Nila Zurin from the laboratory of Dr.Lloyd Green in the Department of Pathology and Cell Biology at Columbia University City. Today we will demonstrate how to isolate and culture primary sympathetic neurons from superior cervical ganglia of newborn red pups. In our lab, we typically use these cells to study apoptosis in response to nerve growth factor withdrawal.So let's get started. We begin the procedure by coating a culture dish with rat tail collagen 24 hours before performing the dissection. Depending on the nature of the experiment, a 24 well or 48 well culture plate is used.Next solutions media and equipment will be prepared. These materials and reagents can be found in the text protocol that accompanies this video. It's important to note that the uridine and five FDU solution be made just before use.Continue preparation by fire. Polishing the glass pasture pipettes for the tation steps. The tip of the glass pipette is held in the flame for a few seconds while rotating to narrow and smooth.The opening fire polishing should be conducted in a sterile cell culture hood. To begin the dissection, collect P zero to P one neonatal rat pups. A neonatal rat pup is quickly wiped with 70%ethanol to reduce contamination.Following decapitation, the head is placed on the dissection pad with the ventral side facing up and the coddle end oriented toward you. The severed trachea should be readily visible. Sterile syringe needles are used to secure the severed head to the dissecting pad in the following manner.Push one pin through the roof of the mouth into the pad and push the second pin through the severed spinal cord into the pad. After moving the dissecting pad under the scope and looking through the oculars, use two pairs of forceps to clear away the skin and fat around the trachea. Taking care not to go too deep, a pair of muscles running almost parallel to each other will be exposed on either side of the trachea.During the dissection, you'll need to irrigate with cold sterile PBS or cold serum free RPMI 1640 medium delivered with a sterile pasture pipette to wash away excess blood. This may be done once or more than once, depending on how fast one works. The forceps are then used to cut and remove the muscle on one side first.Once the muscle is removed, the carotid artery will become visible. This artery runs alongside the trachea and bifurcates into what looks like a Y shaped ribbon. This bifurcation is an important landmark and once it is located, it will be relatively easy to find the superior cervical ganglia.The artery is severed at the most coddle end and lifted up slightly with the forceps. The other end of the artery is kept attached. Once this is done, the superior cervical ganglion will become visible.One should see that it is an almond shaped, glossy looking small mass of tissue loosely attached to the artery at the point of bifurcation, and that it has thread like pre and or post ganglionic connectives. A second pair of forceps is used to gently detach the superior cervical ganglion and place it in a small 35 millimeter culture dish containing cold serum free RPMI 1640 medium. Next, the ganglion is extracted on the other side of the trachea following the same protocol.Once all the ganglia have been dissected out, they're freed as much as possible of extraneous tissue. Looking through the scope, you can notice that the ganglia have attached pieces of carotid artery fat or other tissue forceps are used to tease away these unwanted materials. The pre and or post ganglionic connectives are also trimmed away.Each ganglion has about 10, 000 neurons. Once the neurons are plated, they will regrow their neurons cleaned. Ganglia are then placed into a sterile 15 milliliter conical polypropylene tube containing RPMI 1640 media.Next, the sympathetic neuron culture is established. The ganglia are centrifuge at 200 G for two minutes and the snat is discarded. The ganglia are resuspended in 0.5 to one milliliter of 0.25%tripsin solution, and then placed in a 37 degrees Celsius water bath or incubator for 30 minutes to help dissociate the cells in the ganglia.After 30 minutes, 10 milliliters of complete medium are added in order to dilute out and neutralize the trypsin. The solutions are then centrifuge at 200 G for two minutes. The super agent is then discarded and the ganglia are resuspended in two milliliters of final medium.Using one of the pre-prepared fire polished pipette tips with a wide opening, the cells are further dissociated by tri the ganglia up and down 20 times. The tube is then placed on ice for a few minutes. Next, the pipette is fire polished again to make the opening even narrower.After the pipette cools, the ganglia are triturated 20 times more. The medium will become progressively more cloudy, indicating that the cells are being dissociated. This step is repeated once or twice more depending on how well the cells dissociate.After some time, the ganglia will have dissociated into neurons and no intact ganglia will be visible in the tube. There may be some tissue material that will not dissociate at this point. The tube is left to sit on ice for a few minutes so that any unassociated debris can settle to the bottom.All but about 50 microliters of the supernatant is carefully collected and placed in another tube. 50 microliters is left at the bottom of the tube to prevent transfer of unassociated debris. More of the final medium is added to the tube containing dissociated cells.The volume of final medium added depends on the type of culture plate. One is using to culture the neurons. For example, if a 24 well dish is to be employed, then plate one ganglia per well in 0.5 milliliters of final medium per well.A typical rat litter consists of 12 pups, which would give 24 ganglia. This is enough to seed 1 24 well plate, and the total volume of final medium is 12 milliliters. The use of uridine five FDU in the final medium will prevent the proliferation and promote the death of a small population of glia and other cell types.Present in the culture, cells are fed every 48 hours by replacing two thirds of the medium. With fresh RPMI plus 1%horse serum plus NGF and uridine and five FDU. At first, the culture contains sympathetic neurons that look round with no neurons.Short neurons become visible 24 hours after dissection and progressively become denser and more branched over time. The we've just shown you how to isolate and culture primary sympathetic neurons from superior cervical ganglia of newborn rat tubs. When doing this procedure, it's important to remember to one, take time and care 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. Two, during the dissociation step, be patient and take the time to separate the neurons so that they're not found in large clumps. Once they're seated onto the plates, large clumps will make it difficult.In downstream applications such as transecting or infecting the neurons, immuno staining may also be hindered, and any experiments that require counting the neurons will be difficult to carry out as well. Lastly, it is not necessary to supplement the medium with penicillin streptomycin. Every time the cultures are fed additional penicillin streptomycin, the very first time the cells are plated is sufficient.Do add fresh uridine five FDU to the medium every time the medium is exchanged. Our lab has used sympathetic neuronal culture to study apoptosis in response to nerve growth factor withdrawal. So that's it.Thanks for watching and good luck with your experiments.

protocol-for-culturing-sympathetic-neurons-from-rat-superior-cervical

Research

JoVE Journal

Biology

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Learn to isolate and culture primary sympathetic neurons from rat superior cervical ganglia using enzymatic dissociation and collagen-coated plates. This protocol enables neurobiological studies of autonomic nervous system function in vitro.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

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