Inducing Dendritic Growth in Cultured Sympathetic Neurons

Summary

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.

Abstract

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.

Protocol

1. Preparation of Culture Medium (C2 medium)

1. Add the following, in the order listed, to a sterile, disposable 500 ml Erlenmeyer flask: ....

   Amount	Component	Final Concentration
   190 ml	DMEM (low glucose)	N/A
   10 ml	fatty acid free BSA (20mg/ml in DMEM)	50 μg/ml
   2.8 ml	L-Glutamine	1.4 mM
   4 ml	insulin/transferrin/selenium(100x)

Discussion

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.......

Materials

List of materials used in this article

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