Name: inducing dendritic growth in cultured sympathetic neurons
Uploaded: 2012-03-21T12:00:00
Description: Watch this Scientific Journal Video about Inducing Dendritic Growth in Cultured Sympathetic Neurons at JoVE.com

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 &mu;g/ml</td>
			</tr>
			<t...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+and+dendritic+analysis+of+fascia+dentata+granule+cells+in+human+aging+and+senile+dementia.">Morphometric and dendritic analysis of fascia dentata granule cells in human aging and senile dementia.</a> Brain Res. 402, 217-229 (1987).</li><li>Flood, D. G., Coleman, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+plasticity+in+normal+aging+and+decreased+plasticity+in+Alzheimer's+disease.">Hippocampal plasticity in normal aging and decreased plasticity in Alzheimer's disease.</a> Prog. Brain Res. 83, 435-443 (1990).</li><li>Jagadha, V., Becker, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+pathology:+an+overview+of+Golgi+studies+in+man.">Dendritic pathology: an overview of Golgi studies in man.</a> Can. J. Neurol. Sci. 16, 41-50 (1989).</li><li>Bruckenstein, D. A., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+differentiation+of+embryonic+rat+sympathetic+neurons+in+tissue+culture.+I.+Conditions+under+which+neurons+form+axons+but+not+dendrites.">Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites.</a> Dev. Biol. 128, 324-336 (1988).</li><li>Tropea, M., Johnson, M. I., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+promote+dendritic+development+in+rat+sympathetic+neurons+in+vitro.">Glial cells promote dendritic development in rat sympathetic neurons in vitro.</a> Glia. 1, 380-392 (1988).</li><li>Lein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+extracellular+matrix+and+osteogenic+protein-1+on+the+morphological+differentiation+of+rat+sympathetic+neurons.">The effects of extracellular matrix and osteogenic protein-1 on the morphological differentiation of rat sympathetic neurons.</a> Int. J. Dev. Neurosci. 14, 203-215 (1996).</li><li>Lein, P., Johnson, M., Guo, X., Rueger, D., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+protein-1+induces+dendritic+growth+in+rat+sympathetic+neurons.">Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons.</a> Neuron. 15, 597-605 (1995).</li><li>Lein, P. J., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+and+a+basement+membrane+extract+have+different+effects+on+axonal+and+dendritic+outgrowth+from+embryonic+rat+sympathetic+neurons+in+vitro.">Laminin and a basement membrane extract have different effects on axonal and dendritic outgrowth from embryonic rat sympathetic neurons in vitro.</a> 136, 330-345 (1989).</li><li>Higgins, D., Lein, P. J., Osterhout, D. J., Johnson, M. I., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 177-295 (1991).</li><li>Beck, H. N., Drahushuk, K., Jacoby, D. B., Higgins, D., Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein-5+(BMP-5)+promotes+dendritic+growth+in+cultured+sympathetic+neurons.">Bone morphogenetic protein-5 (BMP-5) promotes dendritic growth in cultured sympathetic neurons.</a> BMC Neurosci. 2, 12-12 (2001).</li><li>Guo, X., Rueger, D., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+protein-1+and+related+bone+morphogenetic+proteins+regulate+dendritic+growth+and+the+expression+of+microtubule-associated+protein-2+in+rat+sympathetic+neurons.">Osteogenic protein-1 and related bone morphogenetic proteins regulate dendritic growth and the expression of microtubule-associated protein-2 in rat sympathetic neurons.</a> Neurosci. Lett. 245, 131-134 (1998).</li><li>Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+induce+dendritic+growth+in+cultured+sympathetic+neurons+by+modulating+the+balance+between+bone+morphogenetic+proteins+(BMPs)+and+BMP+antagonists.">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><li>Higgins, D., Lein, P., Osterhout, D., Johnson, M. I., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 177-205 (1991).</li><li>Bruckenstein, D. A., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+differentiation+of+embryonic+rat+sympathetic+neurons+in+tissue+culture.+II.+Serum+promotes+dendritic+growth.">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 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 border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Bellco</td>
			<td>1943-10012</td>
			<td>12 mm Round Coverslip #1 Thickness, German glass</td>
		</tr>
		<tr>
			<td>Bent tip pipets</td>
			<td>Bellco</td>
			<td>1273-50003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P0899-100MG</td>
			<td>Stock solution 1 mg/ml</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Gibco</td>
			<td>15230</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fine forceps Dumont no. 4 and 5</td>
			<td>Fine Science Tools (FST)</td>
			<td>11252-30 11241-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20-gauge needles</td>
			<td>Becton Dickinson</td>
			<td>305176</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leibovitz's L-15 medium</td>
			<td>Invitrogen</td>
			<td>11415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Worthington</td>
			<td>4176</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche</td>
			<td>04942078001</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Hank's balanced salt solution( -Ca,-Mg)</td>
			<td>Invitrogen</td>
			<td>14185-052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM Low glucose</td>
			<td>Invitrogen</td>
			<td>11885</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Harlan Bioproducts</td>
			<td>5017</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ham F-12 nutrient medium (F-12)</td>
			<td>Invitrogen</td>
			<td>11765</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cytosine arabinoside</td>
			<td>Sigma</td>
			<td>C1768</td>
			<td>&nbsp;</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;D Systems Curis, Inc.</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</td>
			<td>P6148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse anti-rat MAP2 antibody</td>
			<td>Sternberg 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>Prolong Antifade kit Molecular Probes</td>
			<td>P7481</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 1. Materials.

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.

Watch this Scientific Journal Video about Inducing Dendritic Growth in Cultured Sympathetic Neurons at JoVE.com

Inducing Dendritic Growth in Cultured Sympathetic Neurons

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 ...

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