Name: Inducing Dendritic Growth in Cultured Sympathetic Neurons (Video) | JoVE Journal
Uploaded: 2012-03-21T12:00:00.000+00:00
Duration: 9 min 52 s
Description: Learn to culture sympathetic neurons from rat superior cervical ganglia and induce dendritic growth using BMP-7 or Matrigel. This protocol enables precise control over neuronal morphology for developmental neuroscience research.

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5 minutes

This work was supported by funding from the National Institutes of Health (grants R21 NS45037 and R01 ES014901).

1. Preparation of Culture Medium (C2 medium)
<ol>
	<li>Add the following, in the order listed, to a sterile, disposable 500 ml Erlenmeyer flask:
	<table border="1">
		<tbody>
			<tr>
				<td>Amount </td>
				<td>Component </td>
				<td>Final Concentration</td>
			</tr>
			<tr>
				<td>190 ml</td>
				<td>DMEM (low glucose)</td>
				<td>N/A</td>
			</tr>
			<tr>
				<td>10 ml</td>
				<td>fatty acid free BSA (20mg/ml in DMEM)</td>
				<td>50 μg/ml</td>
			</tr>
			<tr>
				<td>2.8 ml</td>
				<td>L-Glutamine</td>
				<td>1.4 mM</td>
			</tr>
			<tr>
				<td>4 ml</td>
				<td>insulin/transferrin/selenium(100x)</td>
				<td>10 μg/ml insulin 5.5 μg/ml transferrin 38.7 nM selenium</td>
			</tr>
			<tr>
				<td>0.4 ml</td>
				<td>NGF (125 μg/ml)</td>
				<td>100 ng/ml</td>
			</tr>
			<tr>
				<td>200 ml</td>
				<td>Ham F-12</td>
				<td>N/A</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>Swirl gently to mix and aliquot 10 ml per tube in 17 x 100 sterile snap cap tubes.</li>
	<li>Store at -80 °C</li>
	<li>Important notes:
	<ul type="disc">
		<li>Thaw NGF immediately prior to usage.</li>
		<li>Before aliquoting the NGF stock solution, precoat the interior walls of the tissue culture plastic serological pipet with protein to minimize sticking of NGF to the plastic. Easiest approach is to Pipette the DMEM mixture containing the BSA several times. Rinse the NGF tube several times with the DMEM mixture.</li>
		<li>Add F-12 last because free cysteine may destabilize disulfide bonds in the insulin.</li>
		<li>Don't freeze in volumes greater than 10 ml because CaPO4 crystals may form</li>
		<li>Do not freeze-thaw C2 medium more than 2 times.</li>
	</ul>
	</li>
</ol>
2. Preparation of Glass Coverslips
<ol>
	<li>We have found that only fine German glass coverslips work consistently in this protocol (see Table 1 for recommended source and catalog number).</li>
	<li>Add 75-100 ml of 70% nitric acid to 300 ml glass specimen bottles with polytetrafluoroethylene (PTFE or Teflon) lid liners (Fisher catalog #09-911-765).</li>
	<li>Wearing gloves, drop coverslips one by one into the nitric acid solution. Add enough coverslips to make a layer no more than 2-3 coverslips thick on the bottom of the jar.</li>
	<li>Gently shake the containers with the coverslips for 6-8 hours at room temperature on an orbital shaker set for no more than 185 rpm.</li>
	<li>Working in a fume hood, carefully decant the acid and dispose according to local regulations. Rinse coverslips once with ultra-pure tissue culture-grade water, then add 75-100 ml water to the container. Coverslips may be rinsed multiple times if the water becomes cloudy. Gently shake at room temperature overnight on an orbital shaker set for no more than 185 rpm.</li>
	<li>Repeat the above sequence of nitric acid/ tissue culture-grade water daily for a total of 3 days.</li>
	<li>Decant the final tissue culture-grade water and add enough acetone to barely cover the coverslips.</li>
	<li>Decant this acetone and dispose properly.</li>
	<li>Transfer coverslips to a 150 mm glass Petri dish. Add enough acetone to barely cover the coverslips. Ensure the coverslips are in a single layer in the dish.</li>
	<li>Allow the acetone to evaporate overnight in a fume hood.</li>
	<li>Bake coverslips in a drying oven at 180 °C for 1.5 hours to sterilize.</li>
</ol>
3. Preparation of Coverslips for Culture
<ol>
	<li>The day before the dissection, add 1 glass coverslip (prepared as described above) to each well of a 24 well tissue culture plate using sterile technique. Add 200 μl of sterile poly-D-lysine solution (100 mg/ml in 0.5 M Tris buffer, pH 8) to each well and store the plate at 4 °C overnight.</li>
	<li>The day of the dissection, and ideally before starting the dissection, aspirate the poly-D-lysine solution from each well and wash each well 5 times with sterile tissue-culture grade water.</li>
	<li>Add 200 μl low-glucose DMEM to each plate and store in 37 °C incubator. Approximately 1 hour prior to plating cells, remove low-glucose DMEM and add 250 μl C2 medium per well. Store plates at 37 °C under 5% CO2 until cells are ready to be plated.</li>
</ol>
4. Dissection and Isolation of the Superior Cervical Ganglion
<ol>
	<li>Dissociated cultures of sympathetic neurons are usually prepared by dissociation of superior cervical ganglia (SCG) harvested from prenatal (embryonic days 19-21) rat pups because there are fewer glial cells. However, this same protocol can be used successfully with early postnatal (1 to 3 days old) rat pups or perinatal mouse pups.</li>
	<li>Euthanize pregnant rat via CO2 inhalation. Shave the abdomen and wash the skin with 70% ethanol. Remove the uterine horns under sterile conditions and place the uterine horns in a sterile 150 mm Petri dish. Avoid damaging the intestines or other internal organs of the dam during the dissection.</li>
	<li>Place the Petri dish with the pups onto a pan of ice to anesthetize the embryonic pups. Dissect the embryos free of the uterine horn and amniotic membranes. Cut the spinal cord of each pup along the midline under the shoulder to euthanize the pups. This also severs the vena cava, which reduces bleeding from the carotid artery during dissection of the SCG. Once dissected free of the uterine horn, place embryos in a sterile Leibovitz's L-15 medium (or any air-buffered medium) supplemented with penicillin and streptomycin.</li>
	<li>Place the embryos on their backs on a 150 mm Petri dish half-filled with Sylgard. Using sterile 20 gauge needles, pin the thorax and the head, gently hyperextending the neck, which facilitates dissection of the SCG.</li>
	<li>Make a midline incision along the neck at the level of the clavicles to reveal the submandibular glands. Remove the glands using fine forceps (no. 4 or no. 5 Dumont) to expose the muscle layer.</li>
	<li>Use the fine forceps to cut the sternocleidomastoid muscle near the clavicle. Then gently lift and incise the omohyoid muscle next to the trachea. This muscle is very thin, so avoid cutting too deeply to avoid tearing the underlying carotid artery and SCG. Once these muscle layers are removed, the carotid artery and the vagus nerve should be clearly visible.</li>
	<li>The SCG lies just below the bifurcation of the carotid artery. Using fine forceps (no. 4 or no. 5), blunt dissect tissue away from both sides of the carotid artery to expose the ganglion. Using 2 fine forceps, grasp the carotid artery just above and below the rostral and caudal ends of the SCG, respectively, to remove the section of the carotid artery lying above the SCG as well as the SCG itself. It is likely that the nodose ganglion, which lies alongside the SCG at this stage of development, will also be removed at this step. The nodose ganglia can be identified by its round shape and the large vagal nerve projecting from it. This lies along one of the branches of the carotid artery. In contrast, the SCG is almond shaped and lies directly under the bifurcation of the carotid artery. Place the dissected tissue into a sterile 35 mm culture dish containing L-15 medium and antibiotics. Remove the SCG from all pups before progressing to the next steps.</li>
	<li>Using fine forceps, gently separate the SCG from the carotid artery and any other tissues removed during the dissection (particularly the vagus nerve and nodose ganglion). Use fine forceps to remove the capsule from the ganglia. This greatly reduces the numbers of nonneuronal cells in culture. Transfer the ganglia to another sterile 35 mm dish containing L-15 medium.</li>
	<li>To dissociate the SCG, remove the L-15 medium and replace with 2 ml Collagenase (final concentration 1 mg/ml)/Dispase (final concentration 5 mg/ml) in calcium and magnesium-free Hank's balanced salt solution. Incubate at 37 °C for 40 minutes.</li>
	<li>Transfer the SCG to a sterile 15 ml conical centrifuge tube and bring the volume up to 10 ml with L-15 supplemented with BSA (final concentration 1-2 mg/ml). Centrifuge at 200 X g at room temperature for 5 minutes. Discard the supernatant and wash once more with L-15 supplemented with BSA.</li>
	<li>After the second wash, remove all L-15 and resuspend the ganglia in ~2 ml of C2 medium. Triturate the cells using a fire-polished bent-tip pipette, make sure the pipette is coated with BSA/L15 before trituration to minimize sticking of cells to the glass. Triturate gently 3-4 times. Let the large clumps settle for about 1 minute and transfer the cell suspension to a 15 ml conical tube. Add more C2 media to the remaining SCG and triturate. After letting the clumps settle, transfer the cell suspension from the second trituration to that collected after the first trituration. Repeat this process several times until the ganglia are mostly dissociated. Discard any remaining clumps after the fourth trituration.</li>
	<li>For cells dissociated from one litter of rat pups (typically 12-16 pups), bring the total volume of medium in the cell suspension to 8-10 ml by adding additional C2 medium. Gently resuspend the cells using a pipette and remove a small aliquot to determine the cell density. Adjust the volume of the cell suspension to achieve 2X the cell density desired in the culture, and then, being sure to maintain the cells in suspension, aliquot 250 ml of cell suspension per well. For morphometric studies of dendritic growth, we typically plate cells at low density (~5 x 104 cells per well) in 24 well plates.</li>
	<li>Our laboratory incubates SCG cultures in glass desiccators rather than directly in a CO2 incubator. We do this because sympathetic neurons grown in serum-free medium perform better with a slightly higher CO2 concentration (approximately 6.5%). In addition we do not use antibiotics in our media since it inhibits neurite outgrowth and maintaining cultures in the desiccators seems to minimize spread of contamination if it develops in a subset of cultures. Using this system, we routinely maintain SCG cultures for up to 8 weeks. Once plated, SCG cultures are placed on the porcelain desiccator plate in glass desiccators containing sterile water in the bottom. Approximately 120 ml of CO2 is injected into the desiccator prior to sealing it shut and placing the entire desiccator into an incubator set for 35.5 °C.</li>
</ol>
5. Feeding and Maintenance of Cultures
<ol>
	<li>Cultures are generally fed 3 times a week, with the first media exchange occurring 24 hours after plating. Using a sterile bent-tip pipette, gently withdraw about half of the media per well. Using a clean sterile bent-tip pipette, replace the volume of medium removed with fresh C2.</li>
	<li>To eliminate non-neuronal cells from the culture, the anti-mitotic agent cytosine-D-arabinofuranoside (ARA-C, final concentration 1-2 μM) is typically added to the culture medium at the first media exchange (e.g., 24 hours after plating) for 48 hours. This concentration of ARA-C is usually sufficient to remove all non-neuronal cells. If additional ARA-C treatment is required, it is recommended that these be done every other feeding to minimize toxic effects on the neurons.</li>
</ol>
6. Inducing Dendritic Growth with Matrigel or BMP-7
<ol>
	<li>To induce dendritic growth, Matrigel or BMP-7 is added to cultures after non-neuronal cells have been eliminated and ARA-C levels in the cultures are significantly reduced. Typically, we add Matrigel or BMP-7 to the medium on day 5 in vitro, but dendritic growth can be induced by Matrigel or BMP-7 added at any time point in vitro. Matrigel or BMP-7 is added to the C2 medium and cultures are fed as described above.</li>
	<li>Matrigel and BMP-induced dendritic growth is time and concentration-dependent. For maximal effects, Matrigel is added to C2 at a final concentration of 50-75 μg/ml; BMP-7 is added to the C2 medium at a final concentration of 50 ng/ml. Matrigel concentrations of greater than 100 μg/ml or BMP-7 concentrations greater than 100 ng/ml have been noted to cause neuronal toxicity. Dendritic processes (as determined by morphological criteria) become apparent approximately 48 hours after initial exposure to Matrigel or BMP-7 at maximally effective concentrations. To induce robust dendritic growth, cultures are fed with BMP-7 containing medium at every feeding. Equivalent results have been obtained using BMPs 2, 4, 5 and 6 at comparable concentrations 20,21.</li>
</ol>
7. Immunocytochemical Analysis of BMP-7-induced Dendritic Growth
<ol>
	<li>When the desired extent of dendritic arborization has been achieved, cultures are fixed using 4% paraformaldehyde in 0.1 M phosphate buffer. The best fixation is achieved by replacing half the medium in the well with 4% paraformaldehyde and repeating this step 3-5 times over 10-15 minutes.</li>
	<li>Rinse cultures by replacing half the 4% paraformaldehyde with phosphate-buffered saline (PBS) and repeating this step three times over 10- 15 minutes.</li>
	<li>Remove all the PBS and add 200 μl 0.1% Triton X-100 in PBS to each well for 5 minutes at room temperature to permeabilize the cells.</li>
	<li>Remove Triton solution by aspiration and add 200 μl per well of blocking buffer solution (PBS supplemented with 3-5% BSA and 1% goat serum). Incubate for 20-30 minutes at room temperature.</li>
	<li>Remove the blocking buffer and add the primary antibody, microtubule-associated protein-2 (MAP2) diluted at 1:2000 - 1:5000 in blocking buffer. Incubate cultures for 1 hour at room temperature or overnight at 4 °C.</li>
	<li>Remove the primary antibody solution, wash the cultures with PBS three times over 15 minutes at room temperature.</li>
	<li>Incubate the cultures with the secondary antibody diluted in PBS supplemented with 1% BSA for 1-2 hours at room temperature in the dark.</li>
	<li>Remove the secondary antibody solution and wash the cultures with PBS three times over 10-15 minutes at room temperature in the dark.</li>
	<li>Mount coverslips on glass slides cell side down in a drop of aqueous mounting medium at the appropriate pH for the fluorochrome tagged to the secondary antibody. Keep the slides at room temperature overnight to let the mounting medium dry. Store slides at 4 °C until imaging.</li>
	<li>Allow the slides to equilibrate to room temp before imaging. Acquire fluorescent images using a phase-contrast microscope equipped for epifluorescence. Typically, the entire dendritic arbor of a single neuron can be captured using a 20X objective and appropriate filters.</li>
</ol>
8. Representative Results
Sympathetic neurons dissociated from the SCG of perinatal rats and grown in the absence of serum and ganglionic glial cells fail to extend MAP2 immunopositive processes (Figure 1), but rather, typically extend only a single axonal process 14,15. Exposure to BMP-7 (Figure 1) or Matrigel induces the formation of numerous processes that meet the morphological, biochemical and functional criteria of dendrites 17,18. The dendrite-promoting activity of either Matrigel or BMP-7 is concentration- and time-dependent 17,18,20. Maximal dendritic growth is observed using concentrations of Matrigel between 50 and 75 μg/ml or BMP-7 between 30 and 100 ng/ml, and half-maximal effects are typically observed at BMP-7 concentrations of ~2 ng/ml. However, significant changes in dendritic growth can be detected with BMP-7 concentrations as low as 300 pg/ml. The dendritic response to either Matrigel or BMP-7 is relatively slow, with &lt;50% of the neurons forming a second process within 24 hours after exposure to either dendrite-promoting agent. However, within 3 days after the initial BMP-7 exposure, virtually all neurons respond to maximally effective Matrigel or BMP-7 concentrations. The number of dendrites per neurons continues to increase with continuous exposure to BMP-7, with most of the change occurring during the first 10 days of treatment. After 4 weeks, BMP-7-treated neurons typically have 6-8 primary dendrites that exhibit secondary, tertiary and even quaternary dendritic branches 17. This increase in dendritic arbor occurs in the absence of any significant change in axonal growth, and comparable dendritic responses can be elicited using similar concentrations of BMPs 2, 4, 5 or 6 20,21. Dendritic growth can also be elicited by co-culturing sympathetic neurons with endogenous ganglionic glial cells 15,22; however, the dendritic response is significantly slower under these conditions.
<img alt="Neuronal differentiation process, microscope image, showing cell morphology changes, 50μm scale." src="/files/ftp_upload/3546/3546fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. BMP-7 promotes dendritic growth in cultured sympathetic neurons. Non-neuronal cells were eliminated from SCG cultures by treatment with anti-mitotic for 48 hr beginning on day 2 in vitro. Beginning on day 5 in vitro, cultures were treated with either control medium (A) or medium supplemented with BMP-7 at 50 ng/ml (B). On day 11 in vitro (after 5 days of BMP treatment), cultures were immunostained with mAb against MAP2, a protein found primarily in dendrites and neuronal somata. As shown in representative fluorescence photomicrographs, neurons grown under control conditions lack dendrites as evidenced by the lack of MAP-2 immunopositive processes dendrites (A); in contrast, neurons exposed to BMP-7 (B) typically have several tapered MAP-2 immunopositive dendrites.

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<li>Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glia+induce+dendritic+growth+in+cultured+sympathetic+neurons+by+modulating+the+balance+between+bone+morphogenetic+proteins+%28BMPs%29+and+BMP+antagonists%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Glia induce dendritic growth in cultured sympathetic neurons by modulating the balance between bone morphogenetic proteins (BMPs) and BMP antagonists.</a> J. Neurosci. 22, 10377-10387 (2002).</li>
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<li>Bruckenstein, D. A., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+differentiation+of+embryonic+rat+sympathetic+neurons+in+tissue+culture.+II.+Serum+promotes+dendritic+growth%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth.</a> Dev. Biol. 128, 337-348 (1988).</li>
</ol>

The authors have nothing to disclose.

The advantages of this model include: (a) the cultures are comprised of a homogenous population of sympathetic neurons devoid of other cell types 17,23; (b) the growth factor requirements of sympathetic neurons are well-established, which allows the use of defined medium, and a variety of defined media, and substrates work well for growing and maintaining sympathetic neurons 23; (c) neurons in these cultures respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of th...

<table><tbody><tr><td>Glass coverslips</td><td>Bellco Glass</td><td>1943-10012</td><td>12 mm Round Coverslip #1 Thickness, German glass</td></tr><tr><td>Bent tip pipets</td><td>Bellco Glass</td><td>1273-50003</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P0899-100MG</td><td>Stock solution 1 mg/ml</td></tr><tr><td>Distilled water</td><td>GIBCO, by Life Technologies</td><td>15230</td><td></td></tr><tr><td>Fine forceps Dumont no. 4 and 5</td><td>Fine Science Tools</td><td>11252-30 11241-30</td><td></td></tr><tr><td>20-gauge needles</td><td>BD Biosciences</td><td>305176</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 medium</td><td>Invitrogen</td><td>11415</td><td></td></tr><tr><td>Collagenase</td><td>Worthington Biochemical</td><td>4176</td><td>1 mg/ml</td></tr><tr><td>Dispase</td><td>Roche Group</td><td>04942078001</td><td>5 mg/ml</td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution( -Ca,-Mg)</td><td>Invitrogen</td><td>14185-052</td><td></td></tr><tr><td>DMEM Low glucose</td><td>Invitrogen</td><td>11885</td><td></td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030</td><td>20 mM</td></tr><tr><td>Insulin/Transferrin/ Selenium</td><td>Invitrogen</td><td>51500-056</td><td>100X</td></tr><tr><td>Fatty-acid free BSA</td><td>Calbiochem</td><td>126609</td><td></td></tr><tr><td>NGF</td><td>Harlan Laboratories</td><td>5017</td><td></td></tr><tr><td>Ham F-12 nutrient medium (F-12)</td><td>Invitrogen</td><td>11765</td><td></td></tr><tr><td>Cytosine arabinoside</td><td>Sigma-Aldrich</td><td>C1768</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>356234</td><td>Avoid repeated freeze-thaw cycles</td></tr><tr><td>BMP-7</td><td>R&amp;amp;D Systems</td><td>345-BP BMP7-07H</td><td>BMPs 4, 5 and 6 work equally as well as BMP-7 and are sometimes easier to obtain commercially</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr><tr><td>Mouse anti-rat MAP2 antibody</td><td>Sternberger Monoclonals Inc.</td><td>SMI52</td><td>1/5000</td></tr><tr><td>Alexa Fluor 546 goat-anti-mouse</td><td>Invitrogen</td><td>A11003</td><td>1/10000</td></tr><tr><td>Mounting media</td><td>Molecular Probes, Life Technologies</td><td>P7481</td><td></td></tr></tbody></table>

inducing dendritic growth in cultured sympathetic neurons

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

The aim of this procedure is to culture sympathetic neurons so as to be able to manipulate their dendritic growth. This is accomplished by first isolating the superior cervical ganglia from the perinatal rodent pups under sterile conditions. The second step is to dissociate the ganglia to form a single cell suspension that is plated on the prepared substrates.Inde defined medium. Then after treating the cultures with anti-mitotic to eliminate the non neuronal cells, treat the cultures with BMPs or matrigel to induce dendritic growth. Ultimately, map two immunoreactivity is used to show the extended dendrites of the cultured sympathetic neurons.The main advantage of this technique over existing methods like primary culture of hippocampal neurons, is that it is possible to manipulate the morphologic state of DYS neurons such that less than 5%of the neurons in culture have dendrites, or more than 95%of the neurons in culture exhibit robust ntic outgrowth To prepare the associated cultures of the SCG from the prenatal rat pups at 19 to 21 embryonic days, first shave the abdomen of a euthanized pregnant rat, clean the skinless 70%ethanol, then remove the uterine horns under sterile conditions and avoid damaging the intestines or other internal organs. Place the uterine horns in a sterile 150 millimeter Petri dish. Next, transfer the Petri dish onto a pan of ice.For anesthetizing the embryonic pups to euthanize the pup, cut the spinal cord along the midline under its shoulder. This also severs the vena caver, which would reduce bleeding from the carotid artery During the dissection of the SCG place the embryos in sterile leibovitz's L 15 medium supplemented with penicillin and streptomycin. Then transfer the embryos to a 150 millimeter Petri dish, half filled with sil guard and place them on their backs.Use the sterile 20 gauge needles to pin the thorax and the head gently hyperextending the neck to facilitate the dissection of the SCG under the microscope. Make a midline incision along the neck at the level of the clavicles to reveal the submandibular glands. Remove the glands using fine forceps in order to expose the muscle layer.Then cut the sternocleidomastoid muscle near the clavicle, gently lifting inci the omohyoid muscle next to the trachea. This muscle is very thin, so avoid cutting too deeply, which would tear the underlying carotid artery and SCG. Once these muscle layers are removed, the carotid artery and the vagus nerve should be clearly visible.The SCG lies just below the bifurcation of the carotid artery blunt. Dissect the tissue away from both sides of the carotid artery with the fine forceps in order to expose the ganglion. Then use two fine forceps to grasp the carotid artery just above and below the rostral and cordal lens of the SCG respectively.To remove the section of the carotid artery lying above the SCG as well as the SCG itself, it is likely that the noose ganglion, which lies alongside the SCG at this stage of development, will also be removed at this step. Place the dissected tissue into a sterile 35 millimeter culture dish containing L 15 medium and antibiotics. After that, remove the SCG from all the pups before progressing to the next steps.Next, gently separate the SCG from the carotid artery and any other tissues. Remove during the dissection. Remove the capsule from the ganglia.Then transfer the ganglia to another sterile 35 millimeter dish containing L 15 medium. To dissociate the SCG remove L 15 medium. Replace it with collagenase dys basing calcium and magnesium free hanks balance sort solution.Then incubate it at 37 degrees Celsius for 40 minutes. After that, transfer the SCG to a sterile 15 milliliter conical centrifuge tube. Bring the volume up to 10 milliliters with L 15 supplemented with BSA to a final concentration of one to two milligrams per milliliter.Centrifuge at 200 times G at room temperature for five minutes. After five minutes, discard the supinate and wash it once more with L 15 supplemented with BSA after the second wash. Remove L 15 medium and rein the ganglia in two milliliters of culture.Medium tri rate the cells about three to four times using a fire polished bent tip pipette. Make sure the pipette is coated with BSAL 15 before TRI to minimize sticking of cells to the glass. Let the large clumps settle for about one minute, then transfer the cell suspension to a 15 milliliter conical.Subsequently add more culture media to the remaining SCG and tri rate. After the clumps have settled, transfer the cell suspension from the second tri to that collected after the first tri. Repeat this process several times until the ganglia are mostly dissociated.Discard any remaining clumps after the fourth tri for the cells dissociated from one liter of rat pups, the total volume of medium in the cell suspension should be brought to eight to 10 milliliters. By adding additional culture medium, gently resuspend the cells using a pipette. Then collect a small aliquot for the determination of cell density.Adjust the volume of the cell suspension in order to achieve two times the cell density desired in the culture. Next, aliquot 250 microliters of cell suspension per well. For the morphometric studies of dendritic growth, we typically plate cells in 24, well plates at low density, about five times 10 of the four cells per well.Then place the SCG cultures on the porcelain desiccate plate in the glass desiccate containing sterile water. Inject approximately 120 milliliters of carbon dioxide into the desiccate prior to sealing it shut. After that, place the entire desiccate into an incubator and set it 35.5 degrees Celsius.In this procedure. Feed the culture by exchanging half of the medium 24 hours after plating and repeat three times a week to eliminate non neuronal cells from the culture. The anti-mitotic agent, RRC as a concentration of one to two micromolar is typically added to the culture medium at this time.Then add the fresh culture medium with RSC to each well and culture for 48 hours to induce dendritic growth after the non neuronal cells have been eliminated and RSC level has been significantly reduced. Add matrigel or BMP seven to the culture medium on day five. In vitro then feed the culture as described previously shown here as a neuron grown under the control conditions with the lack of dendrites as evidenced by the lack of MAP two immuno positive processes.In contrast, this neuron, which has been exposed to BMP seven has several tapered map two immuno positive dendrites shown here. A cultured sympathetic neurons are five to seven days in culture prior to treatment with BMP seven and post-treatment with BMP seven at 30 to 50 nanograms per milliliter for two days. Note, the beginning of dendritic growth.Culture's grown for seven to 14 days in the absence of bmp.Seven. Continue to exhibit only axonal growth while cultures treated with BMP.Seven. At 30 to 50 nanograms per milliliter for seven to 14 days.Exhibit robust dendritic growth. After watching this video, you should have a good understanding of how to isolate SCG. Prepare dissociated culture of sympathetic neurons for the studies of neuronal morphogenesis and experimentally manipulate dendritic outgrowth.

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Research

JoVE Journal

Neuroscience

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

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

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Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

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Inducing Dendritic Growth in Cultured Sympathetic Neurons

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